Methods and kits for detecting misfolded proteins

ABSTRACT

Methods, kits and compounds are provided that relate to the diagnosis, treatment, and/or prevention of preeclampsia.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/679,285, filed Aug. 17, 2017, which is a continuation of U.S.application Ser. No. 14/849,670, filed Sep. 10, 2015, now U.S. Pat. No.10,048,276, which is a continuation of U.S. application Ser. No.13/126,757, filed Aug. 3, 2011, now U.S. Pat. No. 9,229,009, which is anational stage filing under 35 U.S.C. § 371 of international applicationnumber PCT/US2009/005957, filed Nov. 2, 2009, which was published underPCT Article 21(2) in English and claims the benefit under 35 U.S.C. §119(e) of U.S. provisional application No. 61/197,914, filed on Oct. 31,2008, and U.S. provisional application No. 61/206,534, filed Jan. 29,2009, each of which is herein incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

Preeclampsia complicates 6-8% of pregnancies (Hauth, J. C. et al.,Obstet Gynecol. 95(1):24-8, 2000) with an incidence in the US of 23.6cases per 1000 deliveries in the US. (Samadi, A. R. et al., ObstetGynecol. 87(4):557-63, 1996.) Preeclampsia has been determined to beresponsible for 20% of pregnancy-related maternal deaths (MacKay, A. P.et al., Paediatr Perinat Epidemiol. 19(3):206-14, 2005.), the leadingreason for a medically indicated preterm delivery (MIPTD) (Fronterhouse,W. et al., J Matern Fetal Med. 10(3):162-5, 2001.) and responsible for10% of all premature births. (Fronterhouse, W. et al., J Matern FetalMed. 10(3):162-5, 2001.) Preeclampsia (PE) is characterized by the onsetof elevated blood pressure with proteinuria after 20 weeks of gestation.(ACOG Committee on Practice Bulletins. Obstet Gynecol. 99(1):159-67,2002.) It is considered severe (sPE) if blood pressure and proteinuriaare increased substantially or symptoms of end-organ damage, includingfetal growth restriction, occur. The course of severe preeclampsia isassociated with a progressive deterioration of maternal condition andiatrogenic delivery remains the only definitive treatment. Managementfrom the part of the caring physician consists of balancing the risks ofimmediate delivery of an immature fetus against the risks to both motherand child of a complication of preeclampsia. For this, the currentapproach is close monitoring of maternal and fetal status with deliveryremaining the ultimate treatment. (Zamorski, M.A. & Green, L. A. Am FamPhysician 64: 263-70, 216, 2001.)

Currently, there is no single test to predict or diagnose preeclampsiaor to foretell the severity of the condition that will develop in aparticular patient. Early symptoms include persistent headaches, blurredvision or sensitivity to light and abdominal pain. However, a diagnosisof preeclampsia is often not made until increased blood pressure andprotein in the urine (proteinuria) are revealed, typically in routinephysician tests following the 20^(th) week of pregnancy (Roberts J M,Cooper D W. Pathogenesis and genetics of pre-eclampsia. Lancet. 2001;357(9249):53-56). Severe effects of preeclampsia, including seizures,cerebral hemorrhage, disseminated intravascular coagulation and renalfailure, may appear very shortly following such diagnosis. These methodsare imprecise and provide little insight into the likelihood of the mostsevere symptoms developing. Moreover, the current diagnostics requirephysician oversight and invasive methodologies, further delaying andcomplicating early and immediate assessment. An early and accuratemethod for the detection and diagnosis of preeclampsia and associatedproteinuric hypertensive disorders that does not require physicianoversight is needed.

SUMMARY OF THE INVENTION

As described herein, Applicants have shown that preeclampsia (PE) is apregnancy-specific condition characterized by protein misfolding(formation of abnormal or misfolded protein aggregates). In addition,they have shown that characteristic abnormal protein aggregates (alsoreferred to as misfolded protein aggregates or intermediates), arepresent in the urine and accumulate in the placenta of pregnant women(patients) with preeclampsia and are congophilic. These abnormal proteinaggregates are associated with the occurrence of preeclampsia and theirpresence in urine and/or placental tissue is indicative of preeclampsia.As described herein, the term “supramolecular aggregates of misfoldedproteins” (and the shortened term “supramolecular aggregates”)encompasses both soluble protein aggregates and insoluble proteinaggregates. The presence of supramolecular aggregates of misfoldedprotein aggregates that are associated with preeclampsia, such as thosedescribed herein, can be used to determine the presence of preeclampsiain a pregnant woman, as well as the likelihood or risk that a pregnantwoman will develop preeclampsia.

Recent advances in proteomics enabled biomarker discovery and a novelview of preeclampsia (PE) as a misfolding disorder. As described herein,Applicants have determined the presence, as well as the nature andlevel, of protein misfolding in PE. They have shown that PE ischaracterized by increased excretion of misfolded proteins that can bedetected using the affinity of misfolded protein aggregates for certaindyes, such as for example the self-assembling azo dye Congo Red (CR).

Protein conformational disorders, such as Alzheimer's, light chainamyloidosis and prion diseases, are propagated by amyloid fibrilformation and aggregation due to defective folding of cellular proteinsinto aberrant 3D structures. Applicants observed that solublepre-amyloid oligomers (intermediates in fibril assembly, also referredto as misfolded protein intermediates) have proteotoxic effects thatlead to endothelial damage and oxidative stress, which are processesthat play pathogenic roles in severe preeclampsia (sPE). Applicants havefound that the detectable presence of soluble pre-amyloid oligomers isindicative and/or predictive of severe PE and pre-severe PE(pre-preeclampsia), a subclinical state that precedes onset ofclinically manifest sPE.

Thus, Applicants worked to identify and characterize the nature ofurinary soluble pre-amyloid oligomers in this pregnancy-specificcondition. Theirs is the first observation that PE is a conformationaldisorder characterized by amyloid-like assembly of proteins.

Further work described herein, carried out using antibodies thatrecognize proteins and protein oligomers that have adopted a uniquefolding conformation, supports the observation that the misfoldedprotein intermediates have a propensity to assemble into pore-likestructures (amyloid channels) that appear to play a role in clinicaldisease manifestations. The accumulation of abnormal and/or excessivemisfolded protein aggregates in the urine and/or placenta of patientsdiagnosed with PE or destined to develop PE, indicates that theseabnormal protein aggregates are a causative factor in the pathology ofthis disease. This novel finding provides the basis for new diagnosticand therapeutic strategies to treat (reduce, partially or totally, theonset or progression; reverse) preeclampsia. For example, blocking theformation of such structures with immunological or pharmacologicalstrategies and reversing (disrupting) existing supramolecular aggregatesprovide new lines of therapeutic intervention for preeclampsia.

Characteristics of such abnormal protein aggregates (of thesupramolecular amyloid-like assembly of proteins), in addition to theiraffinity for Congo Red, are also described herein. One component ofsupramolecular aggregates abnormal protein aggregates found in urine andplacental tissue in preeclampsia is SerpinA1 (alpha-1 antitrypsin) andpeptide fragments of SerpinA1. Thus, preeclampsia has a characteristicsimilar to that of other disorders, such as alpha-1 antitrypsindeficiency, in which accumulation of misfolded alpha-1 antitrypsin leadsto damage of hepatocytes and cirrhosis of the liver (n. Engl. 1 ed.346:45-53 (2002); J. Clin. Inv. 110: 1585-1590 (2002). In addition,ceruloplasmin, heavy-chain IgG and light-chain IgG, interferon-inducibleprotein 6-16 (IFI6-16, G1P3) and fragments of each were identified incongophilic proteinuria of PE as components of the misfolded proteinaggregates. Isolated abnormal protein aggregates that are associatedwith preeclampsia and are present in urine and placental tissue aredescribed herein and, in specific embodiments discussed further herein,such isolated abnormal protein aggregates comprise at least one (a, oneor more) of SerpinA1, ceruloplasmin, heavy-chain IgG, light-chain IgG,interferon-inducible protein 6-16 and fragments of each.

One embodiment of the invention described herein is a noninvasive urinediagnostic and prognostic test or assay that is based on determining thepresence and, optionally, the quantity of abnormal protein aggregatesthat are associated with preeclampsia, described further herein, thatdemonstrate Congo Red affinity (congophilia) and is a measure of_globalprotein misfolding load in pregnancy. Assessment of global proteinmisfolding load by Congo Red Retention (CRR) is a simple diagnostic testfor PE and for prediction of indicated delivery (IND), which is animportant contributor to preterm birth. The diagnostic and prognosticassays are described herein, in some embodiments, with reference toassessing or analyzing a sample (e.g., urine or placental tissue)obtained from a pregnant woman for the presence of abnormal proteinaggregates that are associated with preeclampsia and exhibitcongophilia. However, it is understood that the diagnostic andprognostic assays can be carried out using a wide variety of agents thatpermit assessment or analysis of a sample to determine the presence(and, optionally, the quantity) of abnormal protein aggregates that areassociated with preeclampsia, as described herein, and that congophiliais but one, descriptive property of the misfolded protein aggregates.Other properties are, for example, the composition and conformation ofthe abnormal protein aggregates, which, in turn, define othercharacteristics of the misfolded protein aggregates, such as their size,interactions with other dyes, and binding by antibodies that can be usedin a diagnostic or prognostic method.

Described herein is a method of diagnosing or aiding in diagnosingpreeclampsia in a pregnant woman by detecting a (one, one or more)supramolecular aggregates of misfolded proteins (abnormal proteinaggregates, misfolded protein aggregates, misfolded proteinintermediates, supramolecular amyloid-like assembly of proteins) that isassociated with preeclampsia in urine or placental tissue of thepregnant woman. It should be appreciated that the diagnostic testsdescribed herein can be carried out at any time during pregnancy. Incertain embodiments, the diagnostic tests described herein can becarried in women planning to become pregnant.

Such supramolecular aggregates can be detected in urine, as well as inplacental tissue, using a variety of techniques or methods known tothose of skill in the art. An appropriate technique is one that permitsdetection of the abnormal protein aggregates in urine or placentaltissue at the levels at which they occur in preeclampsia. Suchtechniques can make use of a dye, a small molecule, a fluorescentcompound or other agent (such as an antibody, antibody fragment,antibody mimetic) that binds or otherwise interacts with abnormalprotein aggregates and, as a result labels, flags or otherwise altersthe aggregates so that they are detectable. One result, in this and inother embodiments described herein, is that the supramolecularaggregates/abnormal protein aggregates, as well as the urine orplacental tissue sample, are different from the originally-obtainedurine or placental tissue and its constituents (e.g., samples have beenmodified, such as by addition of reagents used in their analysis, changein temperature or dilution or concentration; abnormal protein aggregatesare labeled, flagged or otherwise altered, e.g., so that they can bedetected). It should be appreciated that if a dye is used, a dye usefulfor the detection of supramolecular aggregates described herein may haveone or more of the following characteristics: a self-assembling dye, anon self-assembling_dye, a heteroaromatic dye, an azo dye, afibril-specific dye and/or another dye. Such dyes may include, but arenot limited to Congo Red, curcumin analogs; X-34(1,4-bis(3-carboxy-4-hydroxyphenylethenyl)-benzene); Thioflavin S;Thioflavin T; Nile Red; acridine orange; Amino-8-napthalene sulfonate(ANS); Bis-ANS; 4-(dicyanovinyl)-julolidine (DCVJ); A01987 (oxazinedye); fluorescent styryl dyes; BF-168:(6-2-Fluoroethoxy)-2-[2-(4-methylaminophenil)ethenyl]benzoxazole; BSB:(trans,trans)-1-bromo-2,5-bis-(3-hydroxycarbonyl-4-hydroxy)styrylbenzene;quinilinehydrazone compounds (e.g.,4-methyl-7-methoxy-2-(4-quinolylmethylenehydrazino)quinoline);

Chrysamine-G; rifampicin; melatonin; baicalein; scyllitol (e.g.Cocositol, Quercinitol, 1,3,5/2,4,6-Hexahydroxycyclohexane) andderivatives; imaging probes, such as [11C]-PIB: N-methyl [11C]2-(4′-methylaminophenyl-6-hydroxybenzathiazole); stilbenylbenzothiazoleand stilbenylbenzothiazole derivatives.

Self-assembling dyes include for example, Congo Red, Evans Blue, bis-azoANS and non self-assembling dyes include, for example, ANS and TrypanBlue.

As described herein, such abnormal protein aggregates exhibit affinityfor the self-assembling azo dye Congo Red and, thus, their presence inurine or placental tissue can be determined (and, optionally,quantified) by detecting congophilia in urine or placental tissueobtained from a pregnant woman to be assessed (tested) for preeclampsia.Described herein is a method of diagnosing or aiding in diagnosingpreeclampsia in a pregnant woman, in which detection of urinecongophilia, such as by dot blot fixation and spectral shift assays, isindicative of a pregnant woman with preeclampsia (indicates that thepregnant woman has preeclampsia) or as likely to have preeclampsia(which can be confirmed, if needed, by further assessment, using knownmethods). As further described herein, although the abnormal protein isreferred to as congophilic, this is a descriptive term and it is to beunderstood that the abnormal protein can be detected by a variety ofother means, including other agents, such as other dyes (e.g., otherself-assembling dyes), a Thioflavin (Thioflavin S, Thioflavin T) andantibodies (e.g., both polyclonal, such as A11 antibody, Officerantibody, OC antibody and monoclonal, such as M118, M204, M205, M89(such as M89-17) and M09) or by visualizing the aggregates by e.g.polarizing light microscopy, fluorescence microscopy or electronmicroscopy.

The abnormal protein aggregate or fragment thereof detected throughdetection of congophilia can be the abnormal protein aggregate describedherein, which comprises at least one (a, one or more) of SerpinA1,ceruloplasmin, heavy-chain IgG, light-chain IgG, andinterferon-inducible protein 6-16, or a fragment of any of theforegoing.

It should be understood that in all embodiments described herein, avariety of antibodies (polyclonal or monoclonal) can be used. Inspecific embodiments, at least one (a, one or more) of the followingantibodies or their equivalents (antibodies that recognize (bind) insubstantially the same manner (e.g., to the same epitope orconformations can be used: A11 antibody, OC antibody, Officer antibody,M118 antibody, M204 antibody, M205 antibody, M89 antibody (e.g., 89-17)and M09 antibody.

In specific embodiments, the method of diagnosing or aiding indiagnosing preeclampsia in a pregnant woman comprises: (a) obtaining aurine sample or placental tissue sample from the woman; (b) combining(contacting) the sample obtained in (a) with an agent (e.g., a dye, suchas Congo Red, or an antibody) that binds or otherwise labels abnormalprotein aggregate that is associated with PE or binds or otherwiselabels a component of such abnormal protein aggregate, under conditionsunder which binding to or other interaction with abnormal proteinaggregate occurs, thereby producing detectable (labeled) abnormalprotein aggregates or components thereof (e.g., detectable serpina-2 orother component) and (b) determining if binding or other interactionoccurred in (a), such as by determining whether detectable abnormalprotein aggregates or components thereof are in the sample, whereinbinding is indicative of preeclampsia in the woman and woman isdiagnosed as having or likely to have preeclampsia. Her status (havingpreeclampsia or not having preeclampsia) can be further assessed, usingknown methods, such as methods described herein, in order to confirm thedetermination resulting from the present method. The agent that binds orotherwise flags or labels abnormal protein can be any of many typesavailable, such as a dye (e.g., Congo Red, Thioflavin S, Thioflavin T,Evans Blue, Trypan blue, ANS, bis-azo ANS), a radioactive tracer (e.g.(11)C Pittsburgh compound B (PIB)) or an antibody against (that binds)abnormal protein aggregates, such as antibodies that bind acharacteristic region or feature of the conformation or one or more ofthe components of the abnormal protein aggregates, such as one or moreof SerpinA1, ceruloplasmin, heavy-chain IgG, light-chain IgG,interferon-inducible protein 6-16 and fragments of each.

In a specific embodiment, the method of diagnosing or aiding indiagnosing preeclampsia in a pregnant woman comprises: (a) obtaining aurine sample or placental tissue sample from the pregnant woman; (b)combining the sample obtained in (a) with a dye that stains at least oneprotein or protein fragment present in the abnormal protein aggregates(e.g., Congo Red), under conditions under which the dye stains proteinin the sample, thereby producing a urine sample or a placental tissuesample that further comprises the dye; and (c) analyzing the urinesample or placental tissue sample produced in (b) for the presence of(determining if the sample contains) supramolecular aggregates,(abnormal protein aggregates) associated with preeclampsia stained withthe dye, wherein if the sample contains supramolecular aggregatesassociated with preeclampsia stained with the dye, it is indicative ofpreeclampsia in the woman and the woman is diagnosed as having or likelyto have preeclampsia. If the sample contains such stained misfoldedprotein aggregates, she is more likely to have preeclampsia than if thesample does not contain abnormal protein aggregates associated withpreeclampsia stained with the dye. The presence of abnormal proteinaggregates associated with preeclampsia stained with the dye indicatesthat the woman has preeclampsia or is likely to have preeclampsia. Herstatus (having preeclampsia or not having preeclampsia (can be furtherassessed, using known methods, such as methods described herein, inorder to confirm the determination resulting from the present method.The abnormal protein aggregate detected can be the abnormal proteinaggregate described herein, which comprises at least one (one or more)of SerpinA1, ceruloplasmin, heavy-chain IgG, light-chain IgG, interferoninducible protein 6-16 or a fragment of any of the foregoing. Detectionof stained abnormal protein aggregates can be carried out using knownmethods, such as dot blot analysis or simple visualization of an area ofa stained surface (e.g., on a surface, such as filter paper) thatcomprises the urine or placental tissue sample combined with the dye(e.g., urine combined with Congo Red).

A pregnant woman determined, using any of the embodiments describedherein, as having, likely to have or to be at risk of developingpreeclampsia can be further assessed for this condition or the presenceor risk can be confirmed by carrying out an additional assessment, suchas one or more of those described herein or known to one of skill in theart (e.g., blood pressure measurement, assessment of edema, abdominalpain, occurrence of headaches, vision problems).

Detection of urinary congophilic abnormal protein aggregates, asdescribed herein, is not only diagnostic of existing preeclampsia, butalso predictive of the future development of preeclampsia in a pregnantwoman, which can also be seen as predictive of the increased risk that apregnant woman will develop preeclampsia. In one embodiment, the methodof predicting or aiding in predicting the likelihood that a pregnantwoman will have (will develop) preeclampsia comprises obtaining a urineor placental tissue sample from the pregnant woman and analyzing theurine or placental tissue sample for the presence of (determining if thesample contains) abnormal protein aggregates associated withpreeclampsia, wherein if the sample contains abnormal protein aggregatesassociated with preeclampsia, the woman is more likely to develop (hasan increased likelihood of developing) preeclampsia than if the sampledoes not contain abnormal protein aggregates associated withpreeclampsia. It should be appreciated that the methods for assessingthe occurrence and/or risk of PE described herein include methods thatdetect the presence or absence of abnormal protein aggregates associatedwith preeclampsia (e.g. for rapid qualitative assessment) and methodsthat quantify the amount of and/or the precise nature of (e.g.identification of proteins in the aggregate, identification of theconformation of the aggregate, etc.) the supramolecular aggregatesdetected in the sample.

The abnormal protein aggregate detected can be a (one, one or more)supramolecular aggregate described herein, which comprises at least one(a, one or more) of SerpinA1, ceruloplasmin, heavy-chain IgG,light-chain IgG, interferon inducible protein 6-16 or a fragment of anyof the foregoing. The method of predicting whether a pregnant woman willdevelop preeclampsia can be carried out, for example, by (a) obtaining aurine sample or a placental tissue sample from the pregnant woman; (b)combining the urine sample or placental tissue sample with an agent thatbinds or otherwise interacts with misfolded protein aggregatesassociated with preeclampsia, under conditions under which the agentbinds or otherwise interacts with such misfolded protein aggregates or acomponent thereof, thereby producing detectable (labeled) proteinaggregates or components thereof (e.g., detectable SerpinA1 or othercomponent); and (c) determining if binding or other interaction occurredin (b), such as by determining whether detectable misfolded proteinaggregates or components are present in the sample, wherein ifdetectable misfolded protein aggregates are present, the pregnant womanis more likely to develop (has an increased likelihood of developing)preeclampsia than if detectable protein aggregates are not present. Theagent that binds or otherwise flags or labels abnormal protein can beany of many types available, such as a dye (e.g. Congo Red, ThioflavinS, Thioflavin T, Evans Blue, Trypan blue, ANS, bis-azo ANS), aradioactive tracer (e.g. (11)C Pittsburgh compound B (PIB)) or anantibody against (that binds) abnormal protein aggregates, such asantibodies that bind a characteristic region or feature of theconformation or one or more of the components of the aggregates, such asone or more of SerpinA1, ceruloplasmin, heavy-chain IgG, light-chainIgG, interferon-inducible protein 6-16 and fragments of one or more ofthe foregoing.

In a specific embodiment, the method of predicting or aiding inpredicting the likelihood that a pregnant woman will have (will develop)preeclampsia comprises: (a) obtaining a urine sample or a placentaltissue sample from the pregnant woman; (b) combining the sample with adye that stains at least one protein or protein fragment present insupramolecular aggregates associated with preeclampsia (e.g., CongoRed), under conditions under which the dye stains proteins in thesample, thereby producing a sample that further comprises the dye; and(c) analyzing the urine sample or placental tissue sample produced in(b) for the presence of (determining if the sample contains)supramolecular aggregates associated with preeclampsia stained with thedye, wherein if the sample contains supramolecular aggregates associatedwith preeclampsia stained with the dye, the woman is more likely todevelop (has an increased likelihood of developing) preeclampsia than ifthe sample does not contain supramolecular aggregates associated withpreeclampsia stained with the dye. Detection of stained aggregates canbe carried out using known methods, such as dot blot analysis or simplevisualization of an area of a stained surface (e.g., on a surface, suchas filter paper) that comprises the urine or placental tissue samplecombined with the dye (e.g., urine combined with Congo Red).

Similarly, the risk that a pregnant woman will develop preeclampsia canbe assessed using the method described herein. In one embodiment, themethod of determining the risk that a pregnant woman will have (willdevelop) preeclampsia comprises obtaining a urine or placental tissuesample from the pregnant woman and analyzing the urine or placentaltissue sample for the presence of (determining if the sample contains)misfolded protein aggregates associated with preeclampsia, wherein ifthe sample contains misfolded protein aggregates associated withpreeclampsia, the woman is at greater risk of developing preeclampsiathan if the sample does not contain misfolded protein aggregatesassociated with preeclampsia. In a specific embodiment, the method ofassessing or determining the risk that a pregnant woman will developpreeclampsia comprises (a) obtaining a urine sample or a placentaltissue sample from a pregnant woman; (b) combining the urine sample orplacental tissue sample with an agent that binds or otherwise interactswith misfolded protein aggregates associated with preeclampsia, underconditions under which the agent binds or otherwise interacts with suchmisfolded protein aggregates, thereby producing detectable (labeled)protein aggregates or components thereof (e.g., detectable serpina-2 orother component) and (c) determining if binding or other interactionoccurred in (b), such as by determining whether detectable misfoldedprotein aggregates or components are present in the sample, wherein ifdetectable misfolded protein aggregates are present, the pregnant womanis at greater risk of developing preeclampsia than if detectable proteinaggregates are not present. The agent that binds or otherwise flags orlabels abnormal protein can be any of many types available, such as adye (e.g. Congo Red, Thioflavin S, Thioflavin T, Evans Blue, Trypanblue, ANS, bis-azo ANS), a radioactive tracer (e.g. (11)C Pittsburghcompound B (PIB)) or an antibody against (that binds) abnormal proteinaggregates, such as antibodies that bind a characteristic region orfeature of the conformation or one or more of the components of theaggregates, such as one or more of SerpinA1, ceruloplasmin, heavy-chainIgG, light-chain IgG, interferon-inducible protein 6-16 and fragments ofeach of the foregoing.

In a specific embodiment, the method of assessing or estimating the riskthat a pregnant woman will have (will develop) preeclampsia comprises:(a) obtaining a urine sample or a placental tissue sample from thepregnant woman; (b) combining the sample with a dye (e.g., Congo Red)that stains at least one protein or protein fragment present inmisfolded protein aggregates associated with preeclampsia, underconditions under which the dye stains proteins in the sample, therebyproducing a sample that further comprises the dye; and (c) analyzing theurine or placental tissue sample produced in (b) for the presence of(determining if the sample contains) misfolded protein aggregatesassociated with preeclampsia stained with the dye, wherein if the samplecontains misfolded protein aggregates associated with preeclampsiastained with the dye, the woman is at greater risk of developing (has anincreased risk of developing) preeclampsia than if the sample does notcontain misfolded protein aggregates associated with preeclampsiastained with the dye. Detection of stained aggregates can be carried outusing known methods, such as dot blot analysis or simple visualizationof an area of a stained surface (e.g., on a surface, such as filterpaper) that comprises the urine or placental tissue sample combined withthe dye (e.g., urine combined with Congo Red).

Placental tissue samples stained with Congo Red also displaycharacteristic features of such protein aggregates and detectingstaining, such as Congo Red staining. Other labeling of supramolecularaggregates in the placenta can also be used in diagnosing or aiding indiagnosing preeclampsia in a pregnant woman; predicting or aiding inpredicting the likelihood that a pregnant woman will have (will develop)preeclampsia; and assessing or estimating the risk that a pregnant womanwill have (will develop) preeclampsia.

In another embodiment described herein, the method is a method ofdiagnosing or aiding in diagnosing preeclampsia in a pregnant woman, inwhich detection of (determination of the presence of) supramolecularaggregates that are associated with preeclampsia in urine or placentaltissue obtained from the pregnant woman is carried out by usingantibodies that recognize (bind) proteins and protein oligomers thathave adopted a unique folding conformation associated with preeclampsia,as described herein, or antibodies that recognize (bind) supramolecularaggregates and/or a protein or peptide constituent of such aggregatesthat are associated with preeclampsia (as a component of/in the contextof such an aggregate). The presence of such supramolecular aggregates isindicative of (is an indication that the pregnant woman has)preeclampsia.

Also described herein is an ex vivo method for diagnosing preeclampsiaor of predicting the future development of preeclampsia in a subject (apregnant woman), comprising the step of detecting abnormal/misfoldedprotein aggregates (supramolecular aggregates) in a sample of urine orplacenta from said subject. In one embodiment, the supramolecularaggregates (abnormal/misfolded protein aggregates) are detected in asample of urine. In a further embodiment, the abnormal/misfolded proteinaggregates are detected in a sample of placenta. In any of the precedingembodiments, the abnormal protein aggregates are detected by Congo Redstaining, such as by dot blot fixation and/or spectral shift assays, orby Thioflavin S staining. In any of the embodiments, theabnormal/misfolded protein aggregates can comprise SerpinA1 (alpha-1antitrypsin) and/or peptide fragments thereof and/or fragments ofceruloplasmin and/or heavy-chain IgG and/or light-chain IgG.

Also described herein is a protein aggregation inhibitor for use in amethod of treating and/or preventing preeclampsia in a pregnant woman.Protein aggregation inhibitors for use in a method of treating and/orpreventing preeclampsia in a pregnant woman include 1) proteinaggregation inhibitors that inhibit the de novo formation ofsupramolecular aggregates; 2) protein inhibitors that reverse theformation of pre-existing supramolecular aggregates; 3) proteininhibitors that inhibit the de novo formation of supramolecular proteinaggregates and reverse or disrupt pre-existing supramolecular proteinaggregates; 4) protein aggregation inhibitors that stabilize the nativeconformation of the protein, thereby decreasing the rate of abnormalfolding and consequent aggregation; 5) protein aggregation inhibitorsof 1) to 4) that inhibit the aggregation of SerpinA1 (alpha-1antitrypsin) and/or peptide fragments thereof; a protein aggregationinhibitor of any one of 1) to 4), which inhibits the aggregation ofSerpinA1 (alpha-1 antitrypsin) and/or peptide fragments thereof and isselected from: (a) trimethylamine N-oxide; (b) trimethylamineN-oxide-related compounds; (c) the FLEAIG peptide; and (d) peptides andderivatives related to the FLEAIG peptide; 6) a peptide inhibitor of anyone of 1) to 5), which is selected from: (a) trimethylamine N-oxide; (b)trimethylamine N-oxide-related compounds; (c) the FLEAIG peptide; and(d) peptides and derivatives related to the FLEAIG peptide; 7) a peptideinhibitor of any one of 1) to 5), which is an anti-beta amyloid agent;8) a peptide inhibitor of 7) wherein the anti-beta amyloid agent isselected from p-Aminophenol, 2-Amino-4-Chlorophenol pentapeptide iAβ5(LPFFD) (Ax-onyx Inc.); AP aggregation inhibitor PPI-1019 (Praecis); theglycosaminoglycan (GAG) mimetic NC-531 (Neurochem); the antibioticClioquinol (Prana Biotechnology Ltd.), the small molecule cyclodextrin,the natural product from the Ginkgo biloba extract EGb 761 (Dr. WillmanSchwabe GmbH & Co); polyphenols; SP-233, a 22R-hydroxycholesterolderivative (Samaritan Pharmaceuticals), Apomorphine, and AP immunization(Elan Pharmaceutical Inc., Acumen's ADDL technology). See also, DrugDiscovery Today: Therapeutic Strategies 2004 Elsevier Ltd 1(1):7-12.

Also described herein is a method for developing and testing therapies(e.g., drugs) that can be used to treat preeclampsia. For example,drugs, including those known or subsequently found to reduce severity ofother misfolding disorders, can be tested. For example, drugs, thathhave been assessed for their effectiveness in treating, for example,Alzheimer's disease, light chain amyloidosis and prion diseases, can beassessed for their effectiveness in treating preeclampsia, includingpreventing its onset, reducing the extent to which preeclampsia occursor reversing existing preeclampsia (e.g., by disrupting, degrading orotherwise interrupting misfolded protein aggregates associated withpreeclampsia, reducing the burden of pre-existing misfolded proteinaggregates). Such agents, as well as agents that have not yet beenassessed for their usefulness in treating diseases of abnormal proteinaggregations, can be tested in order to identify those of use intreating preeclampsia.

Methods of treating preeclampsia in a pregnant woman are also describedherein. In a method of treating preeclampsia, a pregnant woman is givena therapeutically effective amount of, for example, a drug that thatinhibits (partially or completely) de novo formation of proteinaggregates; a drug that reverses (partially or completely) the burden orexistence of preexisting protein aggregates; a drug that stabilizes thenative conformation of these proteins, thereby decreasing the rate ofabnormal folding and consequent aggregation; or a combination of drugs(more than one drug of the same type or mode of action, such as twodrugs that inhibit de novo formation of protein aggregates; more thanone drug which are not of the same type or mode of action, such as adrug that inhibits de novo formation of protein aggregates and a drugthat reduces the burden of preexisting protein aggregates).

As further described herein, the present disclosure encompasses theembodiments described in the following numbered paragraphs:

1. A method of diagnosing or aiding in the diagnosis of preeclampsia ina pregnant woman, comprising:

(a) obtaining a sample from the woman; and

(b) detecting the presence in the sample of a supramolecular aggregateof misfolded proteins that is associated with (is a causative factor inthe pathology of) preeclampsia, wherein if the supramolecular aggregateis present in the sample, the woman has preeclampsia or is more likelyto have preeclampsia than if the supramolecular aggregate is not presentin the sample.

2. A method of diagnosing or aiding in diagnosing preeclampsia in apregnant woman, comprising:

(a) obtaining a sample from the woman;

(b) combining the sample with a reagent that labels a supramolecularaggregate of misfolded proteins that (i) comprises serpina-1 (alpha-1antitrypsin) or a fragment of serpina-1 and (ii) is associated with (acausative factor in the pathology of) preeclampsia, under conditionsappropriate for labeling of the supramolecular aggregate to occur; and

(c) determining if the supramolecular aggregate is present in thesample,

wherein if the protein aggregate is present, the woman has preeclampsiaor is more likely to have preeclampsia than if the supramolecularaggregate is not present in the sample.

3. The method of paragraph, wherein the supramolecular aggregatecomprises serpina-1 (alpha-1 antitrypsin) or a fragment of serpina-1.

4. The method of paragraph 2 or paragraph 3, wherein the supramolecularaggregate further comprises at least one of ceruloplasmin, heavy-chainIgG, light-chain IgG and interferon inducible protein 6-16 (IFI6).

5. The method of any one of paragraphs 1, 3 or 4, wherein the presenceof the supramolecular aggregate is detected by combining the sample witha reagent that detectably labels the supramolecular aggregate.

6. The method of any one of the paragraphs 1 to 5, wherein the sample isa urine sample or a placental tissue sample.

7. The method of any one of paragraphs 1 to 6, wherein the reagent is adye, an antibody or a fluorescent label.

8. The method of paragraph 7, wherein the dye is a heteroaromatic dye.

9. The method of paragraph 8, wherein the dye is Congo red or aThioflavin.

10. The method of any one of paragraphs 7 to 9, wherein the dye stainssupramolecular aggregates and staining of supramolecular aggregates isdetected by dot blot fixation and/or spectral shift assay.

11. A diagnostic assay for preeclampsia in a pregnant woman, wherein theassay is a determination of the presence of congophilic proteinuria inthe woman and a congophilic protein aggregate that comprises serpina-1(alpha-1 antitrypsin) or a fragment of serpina-1 is detected in a urinesample obtained from the pregnant woman.

12. The method of paragraph, wherein the antibody is aconformation-dependent, amino acid sequence independent antibody.

13. The method of paragraph 12, wherein the conformation-dependentantibody recognizes one or more epitopes on one or more supramolecularaggregates of misfolded protein selected from the group consisting ofprefibrillar oligomer, fibrillar oligomer, protofibril and amyloidfibril.

14. The method of paragraph 13, wherein the conformation-dependentantibody recognizes one or more epitopes on a fibrillar oligomer and/oramyloid fibril.

15. The method of paragraph 13, wherein the conformation-dependentantibody binds one or more epitopes on a (ringshaped) protofibril.

16. The method of paragraph 13, wherein the conformation-dependentantibody binds one or more epitopes on a prefibrillar oligomer.

17. The method of paragraph 14, wherein the conformation specificantibody is OC antibody.

18. The method of paragraph 15, wherein the conformation specificantibody is Officer antibody.

19. The method of paragraph 16, wherein the conformation-specificantibody is A11 antibody.

20. The method of paragraph 15 or 18, wherein if theprotofibril-specific antibody is immunoreactive with the sample, thewoman has developed or is likely to develop hemolysis.

21. A method of diagnosing or aiding in diagnosing preeclampsia in apregnant woman, comprising

(a) obtaining a urine sample or a placental tissue sample from thewoman;

(b) contacting the sample with a conformation-dependent antibody thatbinds a supramolecular aggregate of misfolded protein associated withpreeclampsia, wherein if the antibody binds a component of the sample,the woman has preeclampsia or is more likely to have preeclampsia thanif the antibody does not bind a component of the sample.

22. The method of paragraph 21, wherein the component of the sample issupramolecular aggregate that is a prefibrillar oligomer, a fibrillaroligomer, a protofibril or an amyloid fibril,

23. The method of paragraph 22, wherein the conformation-dependentantibody recognizes one or more epitopes on a fibrillar oligomer and/oramyloid fibril.

24. The method of paragraph 22, wherein the conformation-dependentantibody recognizes one or more epitopes on a (ringshaped) protofibril.

25. The method of paragraph 22, wherein the conformation-dependentantibody recognizes one or more epitopes on a prefibrillar oligomer.

26. The method of paragraph 23, wherein the conformation specificantibody is OC antibody.

27. The method of paragraph 24, wherein the conformation specificantibody is Officer antibody.

28. The method of paragraph 25, wherein the conformation-specificantibody is A11 antibody.

29. The method of paragraph 24 or 27, wherein if theprotofibril-specific antibody is immunoreactive with the sample, thewoman has developed or is likely to develop hemolysis.

30. A method of determining whether a pregnant woman is at risk ofdeveloping preeclampsia, comprising:

(a) obtaining a sample from the woman;

(b) detecting the presence in the sample of a supramolecular aggregateof misfolded proteins that is associated with (is a causative factor inthe pathology of) preeclampsia, wherein if the supramolecular aggregateis present in the sample, the woman is at greater risk of developingpreeclampsia than if the protein aggregate is not present in the sample.

31. A method of determining whether a pregnant woman is at risk ofdeveloping preeclampsia, comprising:

(a) obtaining a sample from the woman;

(b) combining the sample with a reagent that labels a supramolecularaggregate of misfolded proteins that (i) comprises serpina-1 (alpha-1antitrypsin) or a fragment of serpina-1 and (ii) is associated with (isa causative factor in the pathology of) preeclampsia, under conditionsappropriate for labeling of the supramolecular aggregate to occur; and

(c) determining if the supramolecular aggregate is present in thesample,

wherein if the supramolecular aggregate is present, the woman is atgreater risk of developing preeclampsia than if the protein aggregate isnot present in the sample.

32. The method of paragraph 30, wherein the supramolecular aggregatecomprises serpina-1 (alpha-1 antitrypsin) or a fragment of serpina-1.

33. The method of paragraph 31 or 32, wherein the supramolecularaggregate further comprises at least one of ceruloplasmin, heavy-chainIgG, light-chain IgG and interferon inducible protein 6-16 (IFI6).

34. The method of any one of paragraphs 30, 32 or 33, wherein thepresence of the supramolecular aggregate is detected by combining thesample with a reagent that detectably labels the supramolecularaggregate.

35. The method of any one of paragraphs 30 to 34, wherein the sample isa urine sample or a placental tissue sample.

36. The method of paragraph 34 or 35, wherein the reagent is a dye, anantibody, or a fluorescent label.

37. The method of paragraph 36, wherein the dye is Congo red or aThioflavin.

38. The method of any one of paragraphs 35 to 37, wherein Congo red dyestains supramolecular aggregates and staining of supramolecularaggregates is detected by dot blot fixation and/or spectral shift assay.

39. A method of reducing the extent to which preeclampsia occurs in apregnant woman, comprising administering to the woman a therapeuticallyeffective amount of an inhibitor of the occurrence [formation] in thewoman of protein aggregates that comprise serpina-1 (alpha-1antitrypsin) or a serpina-1 fragment and are associated with (are acausative factor of) preeclampsia, wherein a therapeutically effectiveamount is an amount sufficient to inhibit (partially or completely)development of supramolecular aggregates, disrupt (partially orcompletely) existing supramolecular aggregates or both.

40. The method of paragraph 39, wherein the inhibitor stabilizes thenative conformation of at least one component of supramolecularaggregates and decreases the extent to which abnormal folding occurs,whereby formation of supramolecular aggregates in the woman occurs to alesser extent than would occur in the absence of the inhibitor.

41. The method of paragraph 39 or 40, wherein the inhibitor is anantibody or antibody fragment that binds a component of the proteinaggregate, a trimethylamine N-oxide, an anti-beta amyloid agent (e.g.,p-aminophenol, 2-amino-4-chlorophenol, a derivative thereof), a smallmolecule inhibitor of fibril formation, protofibril formation oroligomer formation; or a peptide inhibitor of fibril formation,protofibril formation or oligomer formation.

42. An ex vivo method for diagnosing preeclampsia or of predicting thefuture development of preeclampsia in a subject comprising the step ofdetecting abnormal protein aggregates (supramolecular aggregates ofmisfolded proteins) in a sample of urine or placenta from said subject.

43. The method of paragraph 1, wherein the abnormal protein aggregatesare detected in a sample of urine.

44. The method of paragraph 42, wherein the abnormal protein aggregatesare detected in a sample of placenta.

45. The method of any one of paragraphs 42 to 44, wherein said abnormalprotein aggregates are detected by Congo Red staining.

46. The method of paragraph 45, wherein the Congo Red staining comprisesdot blot fixation and spectral shift assays.

47. The method of any one of paragraphs 42 to 44, wherein said abnormalprotein aggregates are detected by Thioflavin S or Thioflavin Astaining.

48. The method of any one of paragraphs 42 to 47, wherein the abnormalprotein aggregates comprise serpina-1 (alpha-1 antitrypsin) and/orpeptide fragments thereof.

49. The method of any one of paragraphs 42 to 48, wherein the abnormalprotein aggregates comprise fragments of ceruloplasmin and heavy- andlight-chain IgG.

50. A protein aggregation inhibitor for use in a method of treatingand/or preventing preeclampsia.

51. The inhibitor of paragraph 50, which inhibits the de novo formationof protein aggregates.

52. The inhibitor of paragraph 50 or paragraph 51, which reverses ordisrupts the formation of pre-existing protein aggregates.

53. The inhibitor of paragraph 50, which stabilizes the nativeconformation of the protein, thereby decreasing the rate of abnormalfolding and consequent aggregation.

54. The inhibitor of any one of paragraphs 50 to 53, which inhibits theaggregation of serpina-1 (alpha-1 antitrypsin) and/or peptide fragmentsthereof. which is selected from: (a) trimethylamine N-oxide; (b)trimethylamine N-oxide-related compounds; (c) the FLEAIG peptide; and(d) peptides and derivatives related to the FLEAIG peptide.

55. The inhibitor of any one of paragraphs 50 to 54, which is selectedfrom: (a) trimethylamine N-oxide; (b) trimethylamine N-oxide-relatedcompounds; (c) the FLEAIG peptide; and (d) peptides and derivativesrelated to the FLEAIG peptide.

56. The inhibitor of any one of paragraphs 50 to 54, which is ananti-beta amyloid agent.

57. The inhibitor of paragraph 56, wherein the anti-beta amyloid agentis selected from p-Aminophenol, and 2-Amino-4-Chlorophenol.

58. The method of claim 7 wherein the antibody is at least one (one ormore) of the following: A11 antibody, OC antibody, Officer antibody,M118 antibody, M204 antibody, M205 antibody, M89 antibody (e.g., 89-17)and M09 antibody.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a photograph of a nitrocellulose membrane before (left) andafter wash (right) following Congo Red Dot Test of urine samples (5μL/spot were applied to nitrocellulose).

FIG. 2 shows a photograph of a nitrocellulose membrane before and afterwash following Congo Red Dot Test of urine samples (33 μg/spot wereapplied to nitrocellulose) of a cross-sectional cohort. Samples withboxed number were from women with manifest severe preeclampsia.

FIG. 3 shows a photograph of a nitrocellulose membrane before and afterwash following Congo Red Dot Test of urine samples (33 μg/spot wereapplied to nitrocellulose) of a longitudinal cohort. Six pregnant womenwere followed with repeat analysis of urine throughout pregnancy. Theboxed samples were from the time of clinically manifest disease. Theboxes corresponding to samples U348i and U348j were postpartum aftermedically indicated delivered for preeclampsia (emergency C-section).

FIG. 4 shows a bar graph of Congo Red Retention (CRR) normalized foramount of protein in women with severe preeclampsia (sPE), chronichypertension (cHTN) and normal pregnant controls (CRL).

FIG. 5 shows ROC curves of Congo Red Retention (CRR) and Congo Redincorporation (CRI) coefficients to (A) diagnose, and (B) predict amandated delivery for preeclampsia (223 urine samples from 114 differentpregnant women).

FIG. 6 shows a diagram correlating Congo Red Retention (CRR) of urineproteins with the presence and severity of preeclampsia as determined bythe abnormal proteomic profile (UPSr).

FIG. 7 shows a diagram correlating Congo Red Retention (CRR) of urineproteins with the ratio indicator uFP: log [sFlt-1/PIGF×100].

FIG. 8 shows methods of separation of misfolded urine proteins inpreeclampsia by (A) Congo red affinity by dot blot, (B) gel filtration,or (C) centrifugation. Boxed samples are from women with clinicallymanifest severe preeclampsia (sPE).

FIG. 9 shows diagrams showing (A) % Congo Red Retention (CRR) of urineproteins of different treatment groups (CRL: control; cHTN: chronichypertension; gHTN: gestational hypertension; mPE: mild preeclampsia;sPE: severe preeclampsia; spPE: superimposed preeclampsia); (B) % CongoRed Retention (CRR) of urine proteins for different outcome groups (IND:indicated delivery for PE); (C) accuracy graphs comparing the accuracyof prediction (sensitivity/100-specificity) of (1) % Congo Red Retention(CRR) of urine proteins, (2) urine ratio of sFLT/P1GF and (3) P/C ratiourine protein/creatine ratio.

FIG. 10 shows a photograph of a western blot of a nitrocellulosemembrane with spotted urine samples (as indicated: urine, blood,cerebrospinal fluid (CSF) and placenta lysates; PE: preeclampsia; CRL:control) probed with three polyclonal antibodies A11, OC, and Officer.

FIG. 11 shows a photograph of a western blot of a nitrocellulosemembrane with spotted urine samples (PE: preeclampsia; CRL: control;cHTN: chronic hypertension) probed with polyclonal antibody A11. Redarrows mark rows where samples are spotted. The film is compared withthe sample application grid and the position of each sample circled inblack.

FIG. 12 shows a photograph of a western blot of a nitrocellulosemembrane with spotted urine samples (PE: preeclampsia; CRL: control)probed with polyclonal antibody A11 (A11, lane 1) and negative control(neg., lane 2) omitting the primary A11 antibody to control fornon-specific binding of the secondary antibody.

FIG. 13 shows a photograph of a western blot of a nitrocellulosemembrane with spotted urine samples (PE: preeclampsia; CRL: control)probed with monoclonal antibodies (M204, M205, M118, M89-17, M09, M55)and polyclonal A11 (A11) and negative control (Neg.) omitting theprimary antibodies to control for non-specific binding of the secondaryantibody.

FIG. 14 shows a graph for the prediction of indicated delivery forpreeclampsia in a longitudinal cohort.

FIG. 15A shows placental sections from three women, two with pretermdelivery for severe preeclampsia (A-F) and another with idiopathicpreterm birth (Control, G-H) stained with Congo Red and examinedmicroscopically in either white light (A, D, G) or polarized light (B,C, E, F, H and I). Panels C and F are higher magnifications (640×) ofthe squared areas in Panels B & E, respectively Panel I: polarized lightimage of a brain section from a patient with Alzheimer's disease thathas been stained with Congo Red in the same conditions.

FIG. 15B shows imaging of Congo Red positive precipitated material inpreeclamptic urine.

FIG. 16 shows urine samples from two women with severe preeclampsia onduplicate reducing SDS PAGE gels. The left panel shows the gel afterstaining of total proteins with Coomassie blue stain (Lanes 1-4). Theright panel shows the immunoreactivity for SerpinA1 of proteinstransferred to nitrocellulose. Molecular weight markers (MW) are shownon each panel.

FIG. 17 shows secondary screening results for anti-APF monoclonals 09and 89.

FIG. 18 shows results of characterization of monoclonals 09 and 89against alpha hemolysin and AB eta.

DETAILED DESCRIPTION OF THE INVENTION

Preeclampsia develops in the second half of pregnancy and is associatedwith significant maternal and fetal morbidity and mortality. Presently,there is no effective screening test to diagnose or assess the risk ofdeveloping this disease and associated hypertensive disorders. As aresult, pregnant women cannot receive effective monitoring or treatmentuntil after complications associated with the disorders, includingincreased blood pressure and proteinuria, have developed. Additionally,pregnant women with little to no risk of developing such disorders mustundergo unnecessary testing for symptoms throughout their pregnancybecause there is no effective means by which caregivers may exclude themfrom risk in the early stages of pregnancy. Further, current testrequire the oversight of a physician.

Described herein are methods and compositions related to the detectionand/or monitoring of PE-associated congophilia and detection of specificconformations of aggregates of misfolded protein associated with orcausative of PE, which are useful in diagnosis and treatment of PE. Alsodescribed herein are additional biomarkers that can be usedindependently in diagnosis and treatment of PE and, in conjunction withmethods and compositions described herein, as biomarkers that can beused to confirm or further assess the status of a pregnant woman as toPE. For example, a woman who has been assessed, using thepresently-described methods that rely on detection of misfolded proteinaggregates, as having preeclampsia or being at increased risk of havingpreeclampsia, can be further assessed by relying on methods in whichadditional biomarkers or indicators that are associated with (areindicators of) preeclampsia are assessed. This can include, for example,detecting the presence or absence of certain additional biomarkers thatare associated with (indicators of) preeclampsia (e.g. SerpinA1 andalbumin) in a urine sample obtained from the pregnant woman and/ordetermining the ratio of biomarkers that are associated with (areindicators of) preeclampsia (e.g., sFlt-1 and P1GF) in a urine sample.Assessment of these biomarkers can confirm or aid in confirming (ornegate or aid in negating) the assessment, resulting from application ofthe presently-described methods, that a pregnant woman has preeclampsiaor is at risk of having preeclampsia. Methods by which such assessmentscan be carried out are described herein and in the referenced patentdocuments (e.g., U.S. application Ser. No. 12/084,004; PCT/US2006/042585and U.S. Publ. No: US-2006-0183175; PCT/US2005/047010, incorporatedherein by reference in their entirety).

The methods disclosed are useful to diagnose or aid in diagnosis ofpregnant women as having or as being at increased risk for developingany of the following hypertensive disorders: preeclampsia, eclampsia,mild preeclampsia, chronic hypertension, EPH gestosis, gestationalhypertension, superimposed preeclampsia (including preeclampsiasuperimposed on chronic hypertension, chronic nephropathy or lupus),HELLP syndrome (hemolysis, elevated liver enzymes, low platelet count)and nephropathy.

The methods described herein may also be used to assess the risk of apregnant woman developing a specific complication of hypertensivedisorders, including preeclampsia. Such complications may includedelivery by caesarean section, increased serum uric acid, increasedsystolic and diastolic blood pressures, dipstick proteinuria, gravidity,fetal weight at delivery, placental abruption, IUGR, hemolysis,thrombocytopenia, elevated liver enzymes and HELLP syndrome (hemolysis,elevated liver enzymes, low platelet count).

1. Congo Red Test/Congophilia for Diagnosis of PE

Protein instability, misfolding and aggregation into supramolecularstructures with affinity for the self-assembling dye Congo red(congophilia) are features common to a growing number progressive humanconditions, such as Alzheimer's, Parkinson's and prion diseases.Affinity for the azo dye Congo Red (CR) is used to detect aberrantamyloidal aggregates in such diseases associated with proteinmisfolding. Applicants have made the novel and unexpected observationthat preeclampsia (PE) is a pregnancy-specific disease associated withprotein misfolding and protein aggregation. Applicants have shown thatPE is characterized by supramolecular amyloid-like assembly of proteinsand congophilic proteinuria, indicating an increased excretion ofmisfolded proteins. Applicants reasoned that congophilia ofPE-associated proteins in the urine provides a diagnostic and/orprognostic test for PE as a measure of global protein misfolding load inpregnancy.

Misfolded proteins associated with PE can be found in “supramolecularaggregate of misfolded proteins.” As used herein, the term“supramolecular aggregate of misfolded proteins” (herein also referredto as “supramolecular aggregate,” “abnormal protein aggregate,”“supramolecular amyloid assembly” and “congophilic protein aggregate”)refers to soluble protein aggregates and insoluble protein aggregates.These include: (1) pre-fibrillar oligomers (also referred to as“pre-amyloid oligomers” and “non-fibrillar misfolded proteinaggregates”) which are soluble and have adopted “amyloid-like”properties; (2) protofibrils; (3) fibrillar oligomers, which aresoluble; and (4) amyloid fibrils, which are insoluble.

Samples, such as e.g., urine samples, obtained from PE patients,patients suspected of having PE or patients at risk of developing PE,may contain relatively few or no insoluble amyloid fibrils, but maycontain any one of, or a mixture of soluble supramolecular aggregates,such as pre-fibrillar oligomers, fibrillar oligomers, and/orprotofibrils.

In some embodiments, methods of diagnosing PE are provided that comprisedetecting supramolecular aggregates in a sample. Supramolecularaggregates may be detected using, for example, a heteroaromatic and/orfibril-specific dye. In some embodiments, the presence of supramolecularaggregates in a sample, e.g., urine sample, may be detected using thecapacity of these supramolecular aggregates to increase/enhancefluorescence of (or causing a spectral shift in) heteroaromatic dyessuch as thioflavins (e.g. thioflavin T, ThT or thioflavin S, ThS) andCongo red (CR) compared with native protein. In some embodiments,methods of diagnosing PE are provided that comprise detecting solublesupramolecular aggregates, such as pre-fibrillar oligomers in a sampleusing a heteroaromatic and/or fibril-specific dye. Without wanting to bebound by any particular theory, it is thought that the addition of theheteroaromatic and/or fibril-specific dye to soluble supramolecularaggregates promotes the formation and precipitation of insolublesupramolecular aggregates from soluble supramolecular aggregates, suchas pre-fibrillar aggregates and/or fibrillar aggregates, allowing easydetection of these aggregates.

Provided herein are methods for diagnosis or aiding in the diagnosis ofpreeclampsia. In certain embodiments, detection of urine congophilia(e.g., by dot blot fixation and/or spectral shift assays) is indicativeof a pregnant woman with preeclampsia. In certain embodiments, thepresence of urinary congophilic protein aggregates, detected asdescribed, is not only diagnostic of an existing preeclampsia, but alsopredictive of the future development of preeclampsia. In certainembodiments, the presence of congophilic protein aggregates in placentaltissue samples stained with Congo Red is used for the diagnosis ofpreeclampsia. It should be appreciated that the presence of proteinaggregates can be detected through the use of a variety of other agentsand methodologies. Such modifications are well within the capabilitiesof one of ordinary skill and do not involve undue experimentation. Suchmodifications to the methods described herein are meant to be part ofthe invention and are included herein.

In addition to dyes such as Congo Red and thioflavins other agents(including derivatives of Congo Red and thioflavins) may also be usedfor detection of supramolecular aggregates described herein. Thesedetection agents include, but are not limited to, curcumin analogs (e.g.J. Am. Chem Soc. 131: 15257 (2009)); X-34(1,4-bis(3-carboxy-4-hydroxyphenylethenyl)-benzene), a highlyfluorescent Congo red (e.g., Ikonomovic et al. Methods in Enzymology 412(2006), “Amyloid, Prions and other protein aggregates”, Part Bpp:123-144) and J. Histochem and Cytochem 48: 1223 (2000); Thioflavin S;Thioflavin T; Nile Red; acridine orange; Amino-8-napthalene sulfonate(ANS) and Bis-ANS; 4-(dicyanovinyl)-julolidine (DCVJ) (e.g. BiophysicalJournal 94 (12): 4867-4879, 2008); A01987 (oxazine dye) (e.g. NatureBiotechnology 23(5) 577 (2005); fluorescent styryl dyes (e.g. AngewandteChemie, International Edition 43 (46) 6331-6335 (2004); BF-168:(6-2-Fluoroethoxy)-2-[2-(4-methylaminophenil)ethenyl]benzoxazole (e.g.J. Neuroscience 24 (10), 2535 (2004); BSB:(trans,trans)-1-bromo-2,5-bis-(3-hydroxycarbonyl-4-hydroxy)styrylbenzene(e.g. Lab. Investigation 83(12) 1751 (2003); quinilinehydrazonecompounds (e.g.,4-methyl-7-methoxy-2-(4-quinolylmethylenehydrazino)quinoline)(e.g.WO2002/024652, Thomas Raub et. al., Chrisamin G et al.Chrysamine-G, a lipophilic analogue of Congo red, inhibits Abeta-induced toxicity in PC12 cells. Life Sci. 1998; 63(20):1807-14);rifampicin (e.g. Tomiyama T et al. J Biol Chem. 1996; 271:6839-6844);melatonin (e.g. Pappolla M et al. J Biol Chem. 1998; 273:7185-7188);baicalein (e.g. Zhu M et al. The flavonoid baicalein inhibitsfibrillation of alpha-synuclein and disaggregates existing fibrils. JBiol Chem. 2004 Jun. 25; 279(26):26846-57); scyllitol (e.g. Cocositol,Quercinitol, 1,3,5/2,4,6-Hexahydroxycyclohexane) and derivatives (e.g.Sun et al. Synthesis of scyllo-inositol derivatives and their effects onamyloid beta peptide aggregation. Bioorg Med Chem. 2008 Aug. 1;16(15):7177-84); imaging probes, such as [11C]-PIB: N-methyl [11C]2-(4′-methylaminophenyl-6-hydroxybenzathiazole)(e.g. Brain 130: 2607(2007)); stilbenylbenzothiazole and stilbenylbenzothiazole derivatives(e.g. Bioorganic and Medicinal Chemistry Letters 18: 1534 (2008)) andother agents as for example described in FEBS letters 583: 2593 (2009).

In certain embodiments, noninvasive and rapid urine diagnostic assaysare provided. In certain embodiments, these diagnostic assays are basedon Congo red affinity. In certain embodiments, the global proteinmisfolding load of PE patients can be assessed via a simple diagnostictest (e.g., Congo Red retention (CRR) of the urine sample). The CRRtests provided may also be used for prediction of IND, an importantcontributor to preterm birth.

The methods and compositions described herein enable one to assessand/or monitor the risk in a pregnant woman of developing a hypertensivedisorder by detecting and/or monitoring congophilia as measured, forexample, by Congo Red retention. It should be appreciated that otherdyes that interact/associate with protein aggregates and/or dyes thathave chemical properties similar to Congo Red may likewise be used inthe methods described herein and the invention is not limited to the useof Congo Red.

In certain embodiments, diagnostic assays comprise obtaining a proteinsample from a pregnant woman and testing the protein sample forcongophilia. In certain embodiments, the congophilia assay is conductedby contacting the urine sample with the dye Congo Red for sufficienttime to allow Congo Red incorporation (CRI) and then washing the samplewith a wash solution. After washing, Congo Red Retention (CRR) by thesample indicates that the pregnant woman has developed or is at risk ofdeveloping preeclampsia. As references for Congo Red incorporation,positive and negative controls may be incorporated, e.g., from subjectswith diagnosed PE and normotensive subjects, respectively. Theincorporation step and/or the washing step can be controlled, forexample, by including the positive and negative controls, whereinsufficient dye incorporation is indicated when the positive sampleretains Congo Red (is CRR positive) after the washing step, andsufficient washing is indicated when the positive sample does not retainany Congo Red (is CRR negative) after the wash step. In certainembodiments, CRR can be visualized without the need for visualizationequipment, e.g., by separation methods such as, Congo red affinity bydot blot, gel filtration, and centrifugation and washing of theprecipitated Congo red bound proteins with water (see FIG. 8). Incertain embodiments, the separation step may be carried out in a vesselor tube, such as a FALCON™ or EPPENDORF™ tube and dye contacting(retention) and washing can occur within the vessel or tube, followed bya centrifugation step to separate (e.g., precipitate) the dyeincorporated into the protein aggregates from unincorporated dye,optionally followed by additional washing steps. In certain embodiments,the samples may be spotted on a membrane that has affinity to proteins,such as nitrocellulose, and contacting and washing steps may be carriedout on the membrane (e.g. dot blots). Such tests may be performed withonly limited or no supervision by trained physicians, e.g., by trainedtechnicians, or in some embodiments may be performed by thepatient/subject without requiring any supervision and/or laboratoryequipment (such as through the use of a kit that includes the necessaryreagents and instructions).

Applicants have found that although urine samples from a group ofpregnant women vary widely in protein concentration, protein levels inurine are usually sufficiently concentrated to allow for testing ofcongophilia without the need to concentrate the urine sample, allowingfor the design of simple analytic tests that do not require anylaboratory equipment or may require only the most basic equipment.

In certain embodiments, the percent Congo Red Incorporation (% CRI) maybe determined, such as by measuring optical density (OD, densitometry),compared to a negative control and multiplied by the factor 100. Afterwashing the percent Congo Red Retention (% CRR) may be determined bymeasuring optical density (OD) as compared to a negative control andmultiplied by the factor 100. The CRR value of the sample may then besubtracted from the corresponding value of the negative control.Sufficient washing is indicated if no residual Congo Red stain isvisible in the negative control. An OD average should be 0 (if multiplecontrols are used).

In certain embodiments, Congo Red Retention (CRR) and Congo Redincorporation (CRI) coefficients may be calculated and samples withCRR>20% may be “called” Red Dot Test positive and those CRR<15% Red DotTest negative.

In certain embodiments, using the diagnostic assays described herein toidentify the presence of congophilic urine proteins makes it possible toidentify women who develop severe preeclampsia (sPE) 8-10 weeks prior toclinical manifestations. In certain embodiments, detection of urinecongophilia (e.g., by dot blot fixation and/or spectral shift assays) isindicative of a patient with preeclampsia and provides a method fordiagnosis of preeclampsia. In certain embodiments, the presence ofurinary congophilic protein aggregates, detected as described, is notonly diagnostic of an existing preeclampsia, but also predictive of thefuture development of preeclampsia.

In certain embodiments, urine samples may be analyzed immediately aftercollection (e.g., on a dot blot, dipstick or similar) or at a latertime. For example, the urine samples may be frozen at −70° C. and/orcollected in a tube or vessel and dried and stored at e.g. −20° C., or4° C. Drying of the sample may be conducted using a centrifuge undervacuum (e.g., a SPEEDVAC®) and allows for storage and later analysislimiting the risk of sample spoiling (e.g. protein denaturation) attemperatures above freezing. It should be appreciated that drying of thesample to concentrate the sample is usually not necessary, but thesamples may nevertheless be dried for reasons described herein or anyother reason.

2. Protein Aggregate Conformation-Specific Antibodies for Diagnosis ofPE

Applicants have unexpectedly found that PE is a conformational disorderassociated with protein misfolding and aggregation, characterized byamyloid-like assembly of proteins.

Provided herein, in certain embodiments, are methods for diagnosis ofpreeclampsia whereby detection of protein aggregates in the urine ofpregnant women using antibodies that recognize certain proteins and/orprotein oligomers that have adopted a unique folding conformations isindicative of this disease.

Certain embodiments described herein are polyclonal and monoclonalantibodies that may be used to detect protein conformations associatedwith protein aggregation disorders such as PE. Such antibodies, in someembodiments, may include those that detect proteins in e.g. (i) aprefibrillar soluble oligomer conformation, such as the “A11” antibody,provided herein, amongst others, (ii) a ring-shaped protofibrilconformation, such as the “Officer” antibody, provided herein, amongstothers, and (iii) a fibril conformation, such as the “OC” antibody,provided herein amongst others.

As used herein, the term “supramolecular aggregate of misfoldedproteins” (herein also referred to as “supramolecular aggregate,”“abnormal protein aggregate,” “supramolecular amyloid assembly” and“congophilic protein aggregate”) refers to soluble protein aggregatesand insoluble protein aggregates. These include: (1) pre-fibrillaroligomers (also referred to as “pre-amyloid oligomers” and“non-fibrillar misfolded protein aggregates”) which are soluble and haveadopted “amyloid-like” properties; (2) protofibrils; (3) fibrillaroligomers, which are soluble; and (4) amyloid fibrils, which areinsoluble.

Each of these supramolecular aggregates is recognized by antibodies suchas the conformation-dependent, sequence-independent antibodies describedherein.

Protein aggregates with “amyloid-like” properties as used hereindescribe protein aggregates that may share some chemical, physical,biological characteristics with amyloid fibrils but are distinct inother chemical, physical, biological characteristics, such as, forexample for certain amyloid-like protein aggregates, the structure ofthe aggregate.

In some embodiments, pre-fibrillar oligomers are present in the urine ofpatients with PE. In some embodiments, the presence of thesepre-fibrillar oligomers in a sample, e.g. urine sample, may be detectedusing conformation-dependent, amino acid sequence-independentantibodies. In some embodiments, the antibody is A11.

In some embodiments, protofibrils are present in a sample, e.g. a urinesample of a patient with PE. In some embodiments, the presence of theseprotofibrils in a sample, e.g. urine sample, may be detected usingconformation-dependent, amino acid sequence-independent antibodies. Insome embodiments, the antibody is Officer.

In some embodiments, fibrillar oligomers and/or amyloid fibrils arepresent in a sample, e.g., a urine sample of a patient with PE. In someembodiments, the presence of these fibrillar oligomers and/or amyloidfibrils in a sample, e.g., urine sample, may be detected usingconformation-dependent, amino acid sequence-independent antibodies. Insome embodiments, the antibody is OC.

Samples, such as e.g. urine samples, obtained from PE patients orpatients suspected of having PE or patients at risk of developing PE,may contain relatively few or no insoluble amyloid fibrils, but maycontain any one of or a mixture of soluble supramolecular aggregates,such as pre-fibrillar oligomers, fibrillar oligomers, and/orprotofibrils.

Antibodies that recognize supramolecular aggregates in aconformation-dependent, amino acid sequence-independent manner may beraised in animals, such as e.g. mice or rabbit, using preparationscomprising pre-fibrillar aggregates (oligomers), fibrillar aggregates(oligomers), protofibrils or fibrils comprising polypeptides with“amyloid-like” properties that may form such pre-fibrillar aggregates,fibrillar aggregates, protofibrils and/or amyloid fibrils. Peptides with“amyloid-like” properties are known in the art, including amyloid beta(Aβ) peptides, such as e.g. Aβ(1-40) or Aβ(1-42), and polyglutamine(polyGln) molecule NH₂-KKQ₄₂KK—COOH and are described for example inExample 4, Kayed et al. Mol Neurodegener. 2007; 2: 18; Hrncic et al. AmJ Pathol. 2000; 157:1239-1246; O'Nuallain B et al. Proc Natl Acad SciUSA. 2002; 99:1485-1490, references incorporated herein).

Animals can be immunized with morphologically homogeneous populations offibrils, for example to generate conformation-dependent, amino acidsequence-independent antibodies that specifically recognize fibrilsand/or fibrillar aggregates. Some of the resulting antibodies mayspecifically recognize fibrils and/or fibrillar aggregates and do notrecognize (do not cross-react with) random coil monomers, pre-fibrillaroligomers, or natively folded precursor proteins.

Alternatively, animals can be immunized with morphologically amorphouspopulations of pre-fibrillar aggregates, for example to generateconformation-dependent, amino acid sequence-independent antibodies thatspecifically recognize pre-fibrillar aggregates. Some of the resultingantibodies may specifically recognize pre-fibrillar aggregates and donot recognize (do not cross-react with) fibrillar oligomers, amyloidfibrils, monomers or natively folded precursor proteins.

Alternatively, animals can be immunized with populations ofprotofibrils, for example to generate conformation-dependent, amino acidsequence-independent antibodies that specifically recognize ring-shapedprotofibrils. Some of the resulting antibodies may specificallyrecognize protofibrils and do not recognize (do not cross-react with)pre-fibrillar oligomers, fibrillar oligomers, amyloid fibrils, monomersor natively folded precursor proteins.

Applicants have shown that the specificity of detection of PE associatedabnormal protein conformations (supramolecular aggregates) issignificantly improved when the secondary antibody used for detection ispreadsorbed with human IgG to reduce the non-specific binding. This isbased, in part, on the finding by Applicant that PE-associatedaggregated proteins in supramolecular aggregates include humanimmunoglobulins or fragments thereof. Cross-reactivity of a secondaryantibody that has not been pre-adsorbed leads to non-specific binding ofthe secondary antibody. In certain embodiments, methods for detecting PEassociated abnormal protein conformations from urine samples areprovided. Such methods comprise obtaining a urine sample from a pregnantwoman who has or is suspected of having or being at risk of developingPE; contacting the urine sample with a primary antibody (polyclonal ormonoclonal) specific for (that binds) conformations associated withprotein aggregation disorders, under conditions under which misfoldedprotein aggregate-antibody binding occurs, thereby producing acombination; contacting the combination with a secondary antibody thatis preadsorbed with human IgG to reduce non-specific binding, anddetermining if misfolded protein aggregate-antibody complexes arepresent in the urine sample, wherein the presence of misfolded proteinaggregate-antibody complexes (abnormal protein conformations) in theurine sample is indicative of PE.

In certain embodiments, the protein aggregates are contacted with A11primary antibody, described herein, and subsequently with preadsorbedsecondary antibody, wherein A11 positivity in the assay is correlatedwith severity of the PE symptoms.

It should be appreciated that any secondary antibody that is suitablefor detection of the primary antibody can be used as long as thesecondary antibody is sufficiently preadsorbed with human IgG to reduceor prevent non-specific binding and the invention is not limited in thisaspect. The secondary antibody may be labeled with any label to allowdetection, including but not limited to radioisotopic labels, enzymelabels, non-radioactive isotopic labels, fluorescent labels, toxinlabels, affinity labels, chemiluminescent labels and nuclear magneticresonance contrast agents. It should also be appreciated that one ofordinary skill can easily modify or adopt the detection system fordifferent purposes, e.g., high-throughput and/or automated screening,using various labeling and/or detection systems known in the art and theinvention is not limited in this aspect. Further, it should beappreciated that the antibodies described herein are intended to beexamples and one of ordinary skill can generate additional antibodieshaving the characteristics described herein using the methods describedherein and/or methodologies known in the art without the need for undueexperimentation. Antibodies other than those described herein,including, but not limited to, whole antibodies, monoclonal antibodies,polyclonal antibodies, chimeric antibodies, humanized antibodies,primatized antibodies, multi-specific antibodies, single chainantibodies, epitope-binding fragments, fragments comprising either a VLor VH domain, and totally synthetic and recombinant antibodies that havethe characteristics described herein, e.g., that recognize proteinaggregates and/or misfolded or aberrant protein conformations and/orspecific protein fragments (e.g., those of biomarker proteins describedherein) also form part of the invention.

In certain embodiments, monoclonal antibodies are provided thatrecognize protein aggregates and/or misfolded or aberrant proteinconformations. In certain embodiments, monoclonal antibodies areprovided that are raised against the same antigen as the A11 antibody(Science 300: 486-489, 2003), with preferential affinities for differenttypes of prefibrillar protein oligomer conformations. In certainembodiments, monoclonal antibodies #204, #205 and #89 are provided thatshow immunoreactivity for protein aggregates and demonstrate the abilityto detect these protein aggregates in the urine of preeclampsia patientsbut not in controls. In certain embodiments, monoclonal antibodies thatrecognize different conformations of prefibrillar protein oligomers,such as monoclonal #204, #205 and #89 are used in the diagnosis ofpreeclampsia.

Urinary protein aggregates, detected as described, are not onlydiagnostic of an existing preeclampsia, but are also predictive of thefuture development of preeclampsia. In addition to detecting thepresence of protein aggregates in the urine, placental tissue samplescan display characteristic features of such protein aggregates. Thismethodology of detecting abnormally folded proteins and proteinaggregates in the placenta with selective antibodies also hasapplication in the diagnosis of preeclampsia. In certain embodiments,antibodies that bind to protein oligomers are provided that havetherapeutic potential e.g., by (i) disrupting proteinoligomers/aggregates; (ii) preventing further growth of proteinoligomers/aggregates; (iii) stabilizing protein oligomers/aggregates toprevent them from converting into a more pathological confirmation(s);and/or (iv) converting protein oligomers/aggregates from a pathologicalto a non-pathological conformation(s).

Such a therapeutic approaches have been used to treat Alzheimer'sdisease, e.g., using antibodies against the beta-amyloid protein.Applicants have found that preeclampsia is a disease associated with theaccumulation of abnormal protein oligomers. In certain embodiments,antibodies with affinity for unique conformations of abnormally foldedproteins, such as e.g., the monoclonal antibodies #204, #205 and #89-17described herein, or similar, are used as therapeutic agents for thetreatment of preeclampsia.

Applicants have found that protein aggregates present in urine ofsubjects with PE that comprise one or more of immunoglobulin heavy andlight chains, ceruloplasmin and the interferon-inducible protein 6-16(IFI-6) also have therapeutic potential in treating preeclampsia. Incertain embodiments, methods for the diagnosis or prognosis ofpreeclampsia are provided comprising quantitation of one or more ofproteins, such as e.g., immunoglobulin heavy and light chains,ceruloplasmin and the interferon-inducible protein 6-16, in proteinaggregates in the placenta and/or the urine from preeclampsia patients.In certain embodiments, the quantitation of the proteins comprisesrelative protein concentration of these proteins when compared to astandard, e.g., obtained from a normotensive control subject. In certainembodiments, antibodies specific for immunoglobulin heavy and lightchains, ceruloplasmin or the interferon-inducible protein 6-16 are used.

In certain embodiments, antibodies are provided that are specific forconformational epitopes of misfolded protein aggregates found in urinarysamples of patients having, suspected of having or at risk of developingPE and that are useful for the detection of PE. Antibodies providedherein include polyclonal and monoclonal antibodies, as well as antibodyfragments and derivatives that contain the relevant antigen bindingdomain of the antibodies. The term “antibody” as used herein refers toimmunoglobulin molecules or other molecules which comprise at least oneantigen-binding domain. The term “antibody” as used herein is intendedto include whole antibodies (e.g. IgG, IgA, IgE, IgM, or IgD),monoclonal antibodies, polyclonal antibodies, chimeric antibodies,humanized antibodies, primatized antibodies, multi-specific antibodies,single chain antibodies, epitope-binding fragments, e.g., Fab, Fab′ andF(ab′)₂, Fd, Fvs, single-chain Fvs (scFv), disulfide-linked Fvs (sdFv),fragments comprising either a VL or VH domain, and totally synthetic andrecombinant antibodies.

Immunoglobulin or antibody molecules of the invention can be of any type(e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3,IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule.

In certain embodiments, polyclonal antibodies are provided, such ase.g., the “Officer,” “OC,” and “A11” polyclonal antibody. Monoclonal andpolyclonal antibodies can be produced in vivo in response toimmunization with different epitopes on an immunogen, e.g., oligomericaggregates (pre-fibrillar, fibrillar), protofibrillar aggregates oraggregates comprising amyloid fibrils. These aggregates may beconformationally homogenous or heterogeneous/amorphous.

Anti-serum may be raised in a wide range of animals with one or moreinjections of an antigen optionally along with a non-specific enhancerof the immune response, such as an adjuvant. For many small molecules orhaptens, a carrier protein, which may provide determinants recognized byhelper T-cells, may be required for conjugation via variousbi-functional coupling reagents. Homogenous or heterogeneous/amorphousoligomeric aggregates (pre-fibrillar, fibrillar), protofibrillaraggregates or aggregates comprising amyloid fibrils may be administeredwith and adjuvant, e.g., incomplete Freund's adjuvant.

Upon one or more immunizations, the antibodies produced may bepredominantly IgG with some affinity to the epitope. Polyclonalantibodies provide multiple specificity. The specificity of polyclonalantibodies may be improved by affinity chromatography using purifiedantigen.

In certain embodiments, monoclonal antibodies are provided, such ase.g., the “M204,” monoclonal antibody “M205” monoclonal antibody and“M89” monoclonal antibody. Such monoclonal antibodies and others thatrecognize (bind) similar partners, can be used in the methods describedherein, such as of diagnosing or aiding in diagnosing preeclampsia;methods of predictive or aiding in predictive the likelihood a pregnantwoman will develop preeclampsia; and methods of determining or aiding inpredicting whether a pregnant woman is at risk of developingpreeclampsia.

For example, the level of immunoreactivity for monoclonal 89 in urine ofpregnant women is indicative of the risk and severity of HELLP syndrome,an atypical form of severe preeclampsia characterized by Hemolysis,ELevated liver enzymes and Low Platelets. In HELLP syndrome, earlydiagnosis is critical because the morbidity and mortality rates havebeen reported to be as high as 25 percent. The monoclonal antibody hasbeen raised against alpha protofibrils and reacts with those formed byhemolysin which are exotoxins produced by bacteria which cause lysis ofred blood cells in vitro. Results presented in this application (e.g.,FIG. 13) support the use of the relative level of immunoreactivity formonoclonal 89 to that of monoclonal 204, 205 or to polyclonal A11 todetermine the risk for HELLP syndrome and need for urgent intervention.Also described herein is a method of determining the risk or aiding indetermining the risk (likelihood) that a pregnancy woman will developHELLP syndrome and be in need of urgent intervention. The methodcomprises assessing the level of immunoreactivity in a sample (such asurine or placental tissue) for monoclonal 89 relative to that of atleast one of monoclonal 204 or 205 or polyclonal A11. A difference inthe relative levels of immunoreactivity (pregnant woman compared withcontrol, such as relative immunoreactivity in a nonpreeclamptic pregnantwoman or population thereof) are determined and the likelihood apregnant woman will develop HELLP is determined or assisted.

Monoclonal antibodies may be produced in animals such as mice and ratsby immunization. B cells can be isolated from the immunized animal, forexample from the spleen. The isolated B cells can be fused, for examplewith a myeloma cell line, to produce hybridomas, that can be maintainedindefinitely in in vitro cultures. These hybridomas can be isolated bydilution (single cell cloning) and grown into colonies. Individualcolonies can be screened for the production of antibodies of uniformaffinity and specificity. Hybridoma cells may be grown in tissue cultureand antibodies may be isolated from the culture medium. Hybridoma cellsmay also be injected into an animal, such as a mouse, to form tumors invivo (such as peritoneal tumors) that produce antibodies that can beharvested as intraperitoneal fluid (ascites). The lytic complementactivity of serum may be optionally inactivated, for example by heating.

Specific proteins, peptides, haptens, chemical compounds, and proteinaggregates, such as homogenous or heterogeneous/amorphous oligomericaggregates (pre-fibrillar, fibrillar), protofibrillar aggregates oraggregates comprising amyloid fibrils may be used to generateantibodies. One skilled in the art will recognize that the amount ofpolypeptides (e.g. aggregates) used for immunization will vary based ona number of factors, including the animal which is immunized, theantigenicity of the polypeptide selected, and the site of injection. Thepolypeptides (e.g. aggregates) used as an immunogen may be modified asappropriate or administered in an adjuvant in order to increase thepeptide antigenicity. In some embodiments, polypeptides (e.g.aggregates), peptides, haptens, and small compounds may be conjugated toa carrier protein to elicit an immune response. Homogenous orheterogeneous/amorphous oligomeric aggregates (pre-fibrillar,fibrillar), protofibrillar aggregates or aggregates comprising amyloidfibrils may be administered with and adjuvant, e.g. incomplete Freund'sadjuvant.

Suitable methods to increase antigenicity are well known in the art, andinclude, for example, coupling the antigen with a heterologous protein(such as globulin or β-galactosidase) or through the inclusion of anadjuvant during immunization.

Antibody titers can be monitored e.g., by antigen-specific ELISA,western blot analysis, or radioimmunoassay. One or more animals arecommonly used for antibody production. Antibodies or immunospecificfragments thereof of provided herein may be from any animal originincluding rabbits, sheep, goats, chicken, mice, rats, hamsters, guineapigs, donkey, camel, llama, or horse.

After one or more injections of the antigen, approximately 7-10 daysafter each boost, serum may be taken to determine the production ofspecific antibodies (titer). The test bleeds may be assayed against theimmunogen itself, for example in an ELISA assay. Antibodies may bestored in several different buffers, for example at neutral pH, such as0.0 IM phosphate-buffered saline (PBS) at pH 7.4, optionally containing,for example 0.1% sodium azide to inhibit microbial growth. For long-termstorage, antibodies may be kept at a low temperature, such as 4° C.,−20° C. or −70° C. Antibodies may be stored at >0.5 mg/mL and/or in thepresence of a carrier protein (e.g., 1% bovine serum albumin (BSA)), orif frozen, for example in 50% glycerol.

Protocols for generating antibodies, including preparing immunogens,immunization of animals, and collection of antiserum may be found inAntibodies: A Laboratory Manual, E. Harlow and D. Lane, ed., Cold SpringHarbor Laboratory (Cold Spring Harbor, N.Y., 1988) pp. 55-120 and A. M.Campbell, Monoclonal Antibody Technology: Laboratory Techniques inBiochemistry and Molecular Biology, Elsevier Science Publishers,Amsterdam, The Netherlands (1984).

The term “antibody fragment” as used herein is intended to include anyappropriate antibody fragment which comprises an antigen-binding domainthat displays antigen binding function. Antibodies can be fragmentedusing conventional techniques. For example, F(ab′)2 fragments can begenerated by treating the antibody with pepsin. The resulting F(ab′)2fragment can be treated to reduce disulfide bridges to produce Fab¹fragments. Papain digestion can lead to the formation of Fab fragments.Fab, Fab′ and F(ab′)₂, scFv, Fv, dsFv, Fd, dAbs, TandAbs, ds-scFv,dimers, minibodies, diabodies, bispecific antibody fragments and otherfragments can also be synthesized by recombinant techniques or can bechemically synthesized. Techniques for producing antibody fragments arewell known and described in the art. Antibody fragments, includingsingle-chain antibodies, may comprise the variable region(s) alone or incombination with the entirety or a portion of the following: hingeregion, CH1, CH2, and CH3 domains.

In some aspects, the antibody or antibody fragment comprises an antibodylight chain variable region (VL) and an antibody heavy chain variableregion (VH) which generally comprise the antigen binding site. Incertain embodiments, the antibody or antibody fragment comprises all ora portion of a heavy chain constant region, such as an IgG1, IgG2, IgG3,IgG4, IgA1, IgA2, IgE, IgM or IgD constant region. In some aspects, theheavy chain constant region is an IgG1 heavy chain constant region, or aportion thereof. Furthermore, the antibody or antibody fragment maycomprise all or a portion of a kappa light chain constant region or alambda light chain constant region, or a portion thereof. In someaspects, the light chain constant region is a lambda light chainconstant region, or a portion thereof. All or part of such constantregions may be produced naturally or may be wholly or partiallysynthetic. Appropriate sequences for such constant regions are wellknown and documented in the art.

As used herein, the term “heavy chain portion” includes amino acidsequences derived from an immunoglobulin heavy chain. A polypeptidecomprising a heavy chain portion comprises at least one of: a CH1domain, a hinge (e.g., upper, middle, and/or lower hinge region) domain,a CH2 domain, a CH3 domain, or a variant or fragment thereof. Forexample, a binding polypeptide for use in the invention may comprise apolypeptide chain comprising a CH1 domain; a polypeptide chaincomprising a CH1 domain, at least a portion of a hinge domain, and a CH2domain; a polypeptide chain comprising a CH1 domain and a CH3 domain; apolypeptide chain comprising a CH1 domain, at least a portion of a hingedomain, and a CH3 domain, or a polypeptide chain comprising a CH1domain, at least a portion of a hinge domain, a CH2 domain, and a CH3domain. In another embodiment, a polypeptide of the invention comprisesa polypeptide chain comprising a CH3 domain. Further, a bindingpolypeptide for use in the invention may lack at least a portion of aCH2 domain (e.g., all or part of a CH2 domain). It will be understood byone of ordinary skill in the art that these domains (e.g., the heavychain portions) may be modified such that they vary in amino acidsequence from the naturally occurring immunoglobulin molecule.

As used herein, the term “light chain portion” includes amino acidsequences derived from an immunoglobulin light chain. Preferably, thelight chain portion comprises at least one of a VL or CL domain.

In some embodiments, the antibodies or antigen-binding fragments thereofare mammalian antibodies or antigen-binding fragments, such as mouse,rat, rabbit, or human antibodies or antigen-binding fragments.

In a certain embodiments, antibodies of the invention are humanantibodies. The term “human” as used herein in connection with antibodymolecules and fragments thereof refers to antibodies having variable(e.g. VH, VL, CDR or FR regions) and/or constant antibody regionsderived from or corresponding to sequences found in humans, e.g., in thehuman germline or somatic cells.

In some embodiments, human antibodies may be used in human therapy, suchas e.g., in the treatment and/or prevention of PE. In such antibodies,the effector portion may be human and hence it may interact better withthe other parts of the human immune system, that is they are notrecognized by the body as foreign. In certain embodiments, suchantibodies have half-lives similar to naturally-occurring humanantibodies.

In certain embodiments, human antibodies of the invention may alsocomprise one or more amino acid residues which are not naturally encodedby wild-type human nucleic acid sequences, but which have beenartificially changed/introduced in order to modify the sequence of theantibody.

Recombinant techniques are preferred for generating large quantities ofantibodies, antibody fragments and single chain antibodies. In general,recombinant production of antibodies, antibody fragments or derivativesthereof, uses mRNA encoding an antibody which is isolated from hybridomacells that produce the desired antibody. This mRNA is used as a sourcefor generating a cDNA molecule which encodes the antibody, or a fragmentthereof. Once obtained, the cDNA may be amplified and expressedaccording to known methods in a variety of eukaryotic and prokaryotichosts.

In certain embodiments, antibody derivatives are provided. As usedherein, “antibody derivatives” contain an antibody or a fragmentthereof, as well as an additional moiety. Such moieties may improve thesolubility, absorption, biological half-life, etc., of the antibody,decrease the toxicity of the antibody in vivo or in vitro, eliminate orattenuate any undesirable side effect of the antibody in vivo, or serveas a detectable marker of the presence of the antibody. Moieties capableof mediating such effects are well known in the art. In certainembodiments, detectably labeled antibodies are provided. An antibody isreferred to as “detectably labeled” if the antibody, or fragmentthereof, is attached to a molecule which is capable of identification,visualization, or localization using known methods. Suitable detectablelabels include radioisotopic labels, enzyme labels, non-radioactiveisotopic labels, fluorescent labels, toxin labels, affinity labels,chemiluminescent labels and nuclear magnetic resonance contrast agents.

In certain embodiments, hybrid cell lines are provided that secretemonoclonal antibodies selective for specific supramolecular aggregateconformations, such as the specific conformations found in pre-fibrillaroligomers, fibrillar oligomers, ring-shaped protofibrils or amyloidfibrils.

In certain embodiments, these monoclonal antibodies may be used fortreatment and/or prevention of PE, as described herein. These monoclonalantibodies can also be used for qualitative and/or quantitativemeasurement of PE-associated aggregates of misfolded proteins, e.g., inurine samples. These PE-associated aggregates of misfolded proteinsdisplay specific oligomeric conformations that may be detected andmeasured by the monoclonal antibodies provided herein and the presenceof these oligomeric conformations is predictive of the likelihood ofhaving developed PE and/or the risk of developing symptoms of PE.

3. SerpinA1 and Albumin as PE Biomarkers

Applicants have previously demonstrated, using proteomic technology(SELDI-TOF mass spectroscopy) coupled with standard molecular andbiochemical identification assays, that women with preeclampsia havehigher levels of SerpinA1 polypeptides and/or albumin polypeptides intheir urine and other fluids and tissues, than do women withoutpreeclampsia (U.S. application Ser. No. 12/084,004 (PCT/US2006/042585),incorporated herein by reference in their entirety).

In certain aspects, the present invention relates to methods ofdetecting and/or measuring SerpinA1 polypeptides and/or albuminpolypeptides in a sample from a subject (e.g., urine) for determiningpreeclampsia status. In some embodiments, SELDI-based methods ofidentifying subjects with preeclampsia may be used. Such methods mayinclude a step for detecting the level of up to 13 SerpinA1 and albuminpolypeptide biomarkers. The method is based, in part, on a correlationbetween the presence of SerpinA1 and albumin polypeptide biomarkers andthe presence of preeclampsia. Thirteen SerpinA1 and albumin polypeptidebiomarkers have previously been identified (U.S. application Ser. No.12/084,004 (PCT/US2006/042585)), a subset are set forth herein as SEQ IDNOs: 1-6 [MIEQNTKSPLFMGKVVNPTQK (SEQ ID NO:1);M_(ox)IEQNTKSPLFMGKVVNPTQK (SEQ ID NO:2); M_(ox)IEQNTKSPLF_(ox)GKVVNPTQK(SEQ ID NO:3); EDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFS (SEQ ID NO:4);DAHKSEVAHRFKDLGEENFKALVL (SEQ ID NO:5); DAHKSEVAHRFKDLGEENFKALVLIA (SEQID NO:6)]. As used herein, the term “albumin polypeptide” is mean torefer to full-length albumin polypeptide and also refers to apolypeptide that is a fragment of full-length albumin polypeptide.Examples of albumin polypeptide biomarkers are set forth herein as SEQID NOs: 5 and 6 [DAHKSEVAHRFKDLGEENFKALVL (SEQ ID NO:5); andDAHKSEVAHRFKDLGEENFKALVLIA (SEQ ID NO:6)]. The wild-type, full-lengthamino acid sequence of albumin has Genbank Accession No. P02768.

It has been found that the presence in a sample from a patient (pregnantwoman) of a number of the 13 biomarkers that is above certain cut-offvalues indicates the presence of preeclampsia in the pregnant woman. Insuch embodiments of the invention, two objective urinary proteomicscores (UPS) are calculated: a Boolean score (UPSb), which representsthe sum of Boolean indicators assigned to each of the 13 biomarkerscomplemented by a ranked score (UPSr), which retains the quantitativeinformation of the 13 biomarkers with Boolean indicators of 1 (i.e.,objectively present) and is calculated as a rounded integer with thefollowing formula: UPSr=[S/N/10]+1, with S/N=signal to noise from SELDIanalysis. Thus, in theory, the UPSb ranges from 0 to a maximum of 13(one for each of the SerpinA1 and albumin polypeptide biomarkers) andthe UPSr can range from 0 to infinity. Optimum cut-off values as usedfor both UPSb and UPSr to discriminate between subjects with severepreeclampsia and controls without severe preeclampsia. A UPSb levelgreater than six and a UPSr level greater than 8 indicate that thesubject has severe preeclampsia. A UPSb level less than 6 and a UPSrlevel less than 8 indicate that the subject does not have severepreeclampsia. If a sample is determined to have any other combination ofvalues for UPSb and UPSr, a subsequent sample may be obtained from thesubject at a time of 1, 2, 3, 4, 5, 6, or more days after the firstsample was obtained and the second sample may be tested using methods ofthe invention to determine the status of the subject with respect topreeclampsia. Test sample preparation, detection and measurement ofbiomarkers, and diagnosis methods are described herein and also in (U.S.application Ser. No. 12/084,004 (PCT/US2006/042585)).

4. VEGF, P1GF and sFlt-1 as PE Biomarkers

Studies have reported that maternal serum concentrations of vascularendothelial growth factor (VEGF), placental growth factor (P1GF) andsoluble fms-like tyrosine kinase-1 (sFlt-1) are altered in patients withclinical preeclampsia (Levine R J et al. N Engl J Med 2004:12;350:672-83; Maynard S E et al. J Clin Invest 2003; 111:649-58; Levine RJ et al. N Engl J Med 2004: 12; 350: 672-83). Applicants have previouslyshown that endothelial growth factor (VEGF), placental growth factor(P1GF) and soluble fms-like tyrosine kinase-1 (sFlt-1) can serve as abiomarkers for early detection of preeclampsia (U.S. Publ. No:US-2006-0183175; PCT/US2005/047010, incorporated herein by reference intheir entirety). Applicants demonstrated that urinary sFlt-1 issignificantly increased and urinary P1GF is significantly decreased inpregnant women with hypertensive disorders. In certain embodiments, theinvention features methods for measuring the concentration of P1GF andsFlt-1 in a urine sample and utilizing the ratio of such opposing growthfactors to differentiate pregnant women with severe preeclampsia frompregnant women with other forms of hypertensive disorders, includingmild preeclampsia with or without chronic hypertension, or fromnormotensive controls. The methods of the invention may also be used toassess the risk of a pregnant woman developing a specific complicationof hypertensive disorders, including preeclampsia. Such complicationsmay include delivery by caesarean section, increased serum uric acid,increased systolic and diastolic blood pressures, dipstick proteinuria,gravidity, fetal weight at delivery, placental abruption, IUGR,hemolysis, thrombocytopenia, elevated liver enzymes and HELLP syndrome(hemolysis, elevated liver enzymes, low platelet count).

In certain embodiments, a formula is used to analyze results ofdetermination of concentrations or levels of biomarkers. The resultingvalue provides information with respect to the likelihood that thepregnant woman will develop a hypertensive disorder, such aspreeclampsia. As used herein, the term “formula” refers to anymathematical expression, algorithm or other metric that is useful inevaluating whether the levels of an biomarker(s) of interest indicatethat a pregnant woman has or is at risk of developing a hypertensivedisorder and/or specific complications of hypertensive disorders.

In one embodiment, the formula is used to calculate the pregnant woman'suFP. For purposes of this invention, the term “uFP” refers to the log[sFlt-1/PIFG×100]. In one aspect of the invention, a uFP in excess of1.4 is a prognostic indicator of an increased risk that a pregnant womanwill require treatment to prevent the development of or worsening ofsymptoms associated with hypertensive disorders. In another aspect ofthe invention, a uFP in excess of 2.1 indicates that a pregnant womanhas or is at risk for developing severe preeclampsia. In a furtheraspect of the invention, a uFP in excess of 2.1 indicates that apregnant woman is at risk for delivering by caesarean section.

Test sample preparation, detection and measurement of biomarkers, anddiagnosis methods are described herein and also in (U.S. Publ. No:US-2006-0183175; PCT/US2005/047010)

5. Test Sample Preparation (for Certain Embodiments of the Invention)

In certain aspects, a sample from a subject may be a sample collectedfrom a pregnant subject, e.g., a pregnant subject in whom preeclampsiastatus is to be assessed. A pregnant subject may be a pregnant woman whohas been determined to have a high risk of preeclampsia based on herpersonal or family history. A pregnant subject may be a subject who haspreviously been diagnosed with chronic hypertension. Other subjects mayinclude pregnant subjects who are known to have preeclampsia. In someembodiments, the methods of the invention may be used to monitor asubject diagnosed with preeclampsia, for example to determine theeffectiveness of a therapy or treatment administered to the preeclampticsubject. Also, a subjects may be a healthy pregnant woman who is beingtested for preeclampsia as part of a routine examination, or toestablish a baseline level (e.g., a control or reference level) of thebiomarkers in the subject or for other subjects. In other aspects, asample may be collected from a pregnant non-human mammal, or anon-pregnant subject, for example, for use in methods to identify acompound to treat preeclampsia.

Urine samples may be analyzed immediately after collection or at a latertime, provided that, when analyzed, the sample contains detectablelevels of the biomarker(s) of interest. For example, the urine samplesmay be frozen at −70° C. and/or mixed, combined or stored in a containerpretreated with agents that stabilize or preserve the biomarker(s) ofinterest. In a preferred embodiment, the urine sample is collected fromthe first morning void.

Biomarkers of the invention can be measured in different types ofbiological samples, preferably biological fluid samples such as urine.Biomarkers of the invention may also be assessed in tissues and/or inother biological fluid samples. Examples of other biological fluidsamples that may be used in methods and kits of the invention, althoughnot intended to be limiting, include blood, blood serum, plasma, vaginalsecretions, CSF, tears, and saliva. If desired, a sample can be preparedto enhance detectability of the biomarkers. For example, a urine samplefrom the subject can be fractionated. Any method that enriches for abiomarker polypeptide of interest can be used. Sample preparations, suchas prefractionation protocols, are optional and may not be necessary toenhance detectability of biomarkers depending on the methods ofdetection used. For example, sample preparation may be unnecessary if anantibody that specifically binds a biomarker is used to detect thepresence of the biomarker in a sample. Sample preparation may involvefractionation of a sample and collection of fractions determined tocontain the biomarkers. Methods of prefractionation include, forexample, size exclusion chromatography, ion exchange chromatography,heparin chromatography, affinity chromatography, sequential extraction,gel electrophoresis and liquid chromatography. Examples of methods offractionation are described in PCT/US03/00531 (incorporated herein inits entirety).

As an example, a sample is pre-fractionated by anion exchangechromatography. Anion exchange chromatography allows pre-fractionationof the proteins in a sample roughly according to their chargecharacteristics. For example, a Q anion-exchange resin can be used, anda sample can be sequentially eluted with eluants having different pHs.Anion exchange chromatography allows separation of biomolecules in asample that are more negatively charged from other types ofbiomolecules. Proteins that are eluted with an eluant having a high pHis likely to be weakly negatively charged, and a fraction that is elutedwith an eluant having a low pH is likely to be strongly negativelycharged. Thus, in addition to reducing complexity of a sample, anionexchange chromatography separates proteins according to their bindingcharacteristics.

As another example, biomolecules in a sample can be separated byhigh-resolution electrophoresis, e.g., one or two-dimensional gelelectrophoresis. A fraction containing a biomarker can be isolated andfurther analyzed by gas phase ion spectrometry. Preferably,two-dimensional gel electrophoresis is used to generate two-dimensionalarray of spots of biomolecules, including one or more biomarkers. See,e.g., Jungblut and Thiede, Mass Spectr. Rev. 16:145-162 (1997). Thetwo-dimensional gel electrophoresis can be performed using methods knownin the art. See, e.g., Deutscher ed., Methods In Enzymology vol. 182. Incertain cases, biomolecules in a sample are separated by, e.g.,isoelectric focusing, during which biomolecules in a sample areseparated in a pH gradient until they reach a spot where their netcharge is zero (isoelectric point). This first separation step resultsin one-dimensional array of biomolecules. The biomolecules inone-dimensional array is further separated using a technique generallydistinct from that used in the first separation step. Typically,two-dimensional gel electrophoresis can separate chemically differentbiomolecules in the molecular mass range from 1000-200,000 Da withincomplex mixtures. The pI range of these gels is about 3-10 (wide rangegels).

As another example, high performance liquid chromatography (HPLC) canalso be used to separate a mixture of biomolecules in a sample based ontheir different physical properties, such as polarity, charge and size.HPLC instruments typically consist of a reservoir of mobile phase, apump, an injector, a separation column, and a detector. Biomolecules ina sample are separated by injecting an aliquot of the sample onto thecolumn. Different biomolecules in the mixture pass through the column atdifferent rates due to differences in their partitioning behaviorbetween the mobile liquid phase and the stationary phase. A fractionthat corresponds to the molecular weight and/or physical properties ofone or more biomarkers can be collected. The fraction can then beanalyzed by gas phase ion spectrometry to detect biomarkers. Forexample, the spots can be analyzed using either MALDI or SELDI asdescribed herein.

Optionally, a biomarker can be modified before analysis to improve itsresolution or to determine its identity. For example, the biomarkers maybe subject to proteolytic digestion before analysis. Any suitableprotease may be used. Proteases, such as trypsin, that are likely tocleave the biomarkers into a discrete number of fragments areparticularly useful. The fragments that result from digestion mayfunction as a fingerprint for the biomarkers, thereby enabling theirdetection indirectly. This is particularly useful where there arebiomarkers with similar molecular masses that might be confused for thebiomarker in question. Also, proteolytic fragmentation is useful forhigh molecular weight biomarkers because smaller biomarkers are moreeasily resolved by mass spectrometry. Optionally, the identity of thebiomarkers can be further determined by matching the physical andchemical characteristics of the biomarkers in a protein database (e.g.,SwissProt).

In certain embodiments, the invention comprises treating the urinesample(s) from the pregnant woman with one or more stabilizing agentand/or pretreating the container used for collection of such urinesample(s) with one or more stabilizing agent prior to measuring thelevels of angiogenic markers. The term “stabilizing agent” refers to oneor more molecules, such as polypeptides or nucleic acids, that can beused to prevent the degradation of the angiogenic markers. In oneembodiment, the stabilizing agent is a protease inhibitor, including anyof 4-(2-Aminoethyl) benzenesulphonyl fluoride (AEBSF) and Pefabloc SC,Antipain and Antipain-dihydrochloride, Aprotinin, Benzamidine andBenzamidine hydrochloride, Bestatin, Chymostatin, E-64(L-trans-epoxysuccinyl-leucylamide-(4-guanido)-butane orN-[N-(L-trans-carboxyoxiran-2-carbonyl)-L-leucyl]-agmatine),Ethylenediaminetetraacetic acid and its sodium salt (EDTA-Na2),Leupeptin, Ethylmaleimide, Pepstatin and Pepstatin A, Phosphoramidon,Sodium azide, Trypsin inhibitor or ε-aminocaproic acid.

6. Detection and Measurement of Biomarkers (for Certain Embodiments ofthe Invention)

Levels of a biomarker for PE (e.g. SerpinA1 polypeptides and albuminpolypeptides, VEGF polypeptides, sFlt-1 polypeptides, and P1GFpolypeptides) that is useful in a method of the present invention may beassessed by any of a wide variety of well known methods for detectingexpression of a transcribed molecule or its corresponding protein.Non-limiting examples of such methods include immunological methods fordetection of secreted proteins, protein purification methods, proteinfunction or activity assays, nucleic acid hybridization methods, nucleicacid reverse transcription methods, and nucleic acid amplificationmethods. In one embodiment, levels of a bio marker is assessed using anELISA assay.

Methods and compositions described herein permit assessment and/ormonitoring risk in a pregnant woman of developing preeclampsia bydetecting and/or monitoring levels of biomarkers for PE (e.g. SerpinA1polypeptides and albumin polypeptides, VEGF polypeptides, sFlt-1polypeptides, and P1GF polypeptides) in a sample obtained from thepregnant woman. This can be carried out by obtaining a urine sample anddetecting levels of biomarkers of the invention, as described herein, atvaried times as the pregnancy progresses. Resulting values may also becompared to a control or known (pre-established) standard. As usedherein, the terms “appropriate standard” or “control” refers to thelevels of the biomarker in urine obtained from a reference subject. Theappropriate standard concentration can be determined from urine samplesobtained from pregnant women with normal pregnancies or from pregnantwomen who have a confirmed hypertensive disorder, such as preeclampsia(reference or control subjects). In some embodiments of the invention,samples that form the basis of an appropriate standard are obtained fromthe reference subject who, when the sample is obtained, is in the weekof pregnancy corresponding to that week of pregnancy the test subject isin when the test sample is obtained. Samples may be obtained andanalyzed at the same time as urine samples are obtained from testsubjects. Alternatively, PE biomarker levels (e.g. levels of SerpinA1polypeptides and albumin polypeptides, VEGF polypeptides, sFlt-1polypeptides, and P1GF polypeptides) may be determined prospectively orretrospectively to the assessment of the urine sample obtained from atest subject using statistical studies with routine experimentation.Standard PE biomarker levels (e.g., standard levels for SerpinA1polypeptides and albumin polypeptides, VEGF polypeptides, sFlt-1polypeptides, and P1GF polypeptides) can be determined by a personhaving ordinary skill in the art using well known methods.

Biomarkers such as SerpinA1 polypeptides and albumin polypeptides arepreferably captured with capture reagents immobilized to a solidsupport, such as any biochip described herein, a multiwell microtiterplate, a resin, or other suitable support. A preferred massspectrometric technique for use in the invention is Surface EnhancedLaser Desorption and Ionization (SELDI), as described, for example, inU.S. Pat. Nos. 5,719,060 and 6,225,047, in which the surface of a probethat presents the analyte to the energy source plays an active role indesorption/ionization of analyte molecules. In this context, the term“probe” refers to a device adapted to engage a probe interface and topresent an analyte to ionizing energy for ionization and introductioninto a gas phase ion spectrometer, such as a mass spectrometer. A probetypically includes a solid substrate, either flexible or rigid, that hasa sample-presenting surface, on which an analyte is presented to thesource of ionizing energy.

One version of SELDI, called “Surface-Enhanced Affinity Capture” or“SEAC,” involves the use of probes comprised of a chemically selectivesurface (“SELDI probe”). A “chemically selective surface” is one towhich is bound either the adsorbent, also called a “binding moiety,”′ or“capture reagent,” or a reactive moiety that is capable of binding acapture reagent, e.g., through a reaction forming a covalent orcoordinate covalent bond.

The phrase “reactive moiety” here denotes a chemical moiety that iscapable of binding a capture reagent. Epoxide and carbodiimidizole areuseful reactive moieties to covalently bind polypeptide capture reagentssuch as antibodies or cellular receptors. Nitriloacetic acid andiminodiacetic acid are useful reactive moieties that function aschelating agents to bind metal ions that interact noncovalently withhistidine containing peptides. A “reactive surface” is a surface towhich a reactive moiety is bound. An “adsorbent” or “capture reagent”can be any material capable of binding a biomarker of the invention.Suitable adsorbents for use in SELDI, according to the invention, aredescribed in U.S. Pat. No. 6,225,047.

One type of adsorbent is a “chromatographic adsorbent,” which is amaterial typically used in chromatography. Chromatographic adsorbentsinclude, for example, ion exchange materials, metal chelators,immobilized metal chelates, hydrophobic interaction adsorbents,hydrophilic interaction adsorbents, dyes, mixed mode adsorbents (e.g.,hydrophobic attraction/electrostatic repulsion adsorbents). “Biospecificadsorbent” is another category, for adsorbents that contain abiomolecule, e.g., a nucleotide, a nucleic acid molecule, an amino acid,a polypeptide, a simple sugar, a polysaccharide, a fatty acid, a lipid,a steroid or a conjugate of these (e.g., a glycoprotein, a lipoprotein,a glycolipid). In certain instances, the biospecific adsorbent can be amacromolecular structure such as a multiprotein complex, a biologicalmembrane or a virus. Illustrative biospecific adsorbents are antibodies,receptor proteins, and nucleic acids. A biospecific adsorbent typicallyhas higher specificity for a target analyte than a chromatographicadsorbent.

Another version of SELDI is Surface-Enhanced Neat Desorption (SEND),which involves the use of probes comprising energy absorbing moleculesthat are chemically bound to the probe surface (“SEND probe”). Thephrase “Energy absorbing molecules” (EAM) denotes molecules that arecapable of absorbing energy from a laser desorption ionization sourceand, thereafter, contributing to desorption and ionization of analytemolecules in contact therewith. The EAM category includes molecules usedin MALDI, frequently referred to as and is exemplified by cinnamic acidderivatives, sinapinic acid (SPA), cyano-hydroxy-cinnamic acid (CHCA)and dihydroxybenzoic acid, ferulic acid, and hydroxyaceto-phenonederivatives. The category also includes EAMs used in SELDI, asenumerated, for example, by U.S. Pat. No. 5,719,060.

Another version of SELDI, called Surface-Enhanced Photolabile Attachmentand Release (SEPAR), involves the use of probes having moieties attachedto the surface that can covalently bind an analyte, and then release theanalyte through breaking a photolabile bond in the moiety after exposureto light, e.g., to laser light. For instance, see U.S. Pat. No.5,719,060. SEPAR and other forms of SELDI are readily adapted todetecting a biomarker or biomarker profile, pursuant to the presentinvention.

The detection of the biomarkers according to the invention can beenhanced by using certain selectivity conditions, e.g., adsorbents orwashing solutions. The phrase “wash solution” refers to an agent,typically a solution, which is used to affect or modify adsorption of ananalyte to an adsorbent surface and/or to remove unbound materials fromthe surface. The elution characteristics of a wash solution can depend,for example, on pH, ionic strength, hydrophobicity, degree ofchaotropism, detergent strength, and temperature.

In some embodiments of the invention, a sample is analyzed by means of a“biochip,” a term that denotes a solid substrate having a generallyplanar surface, to which a capture reagent (adsorbent) is attached.Frequently, the surface of a biochip comprises a plurality ofaddressable locations, each of which has the capture reagent boundthere. A biochip can be adapted to engage a probe interface and, hence,function as a probe, which can be inserted into a gas phase ionspectrometer, preferably a mass spectrometer. Alternatively, a biochipof the invention can be mounted onto another substrate to form a probethat can be inserted into the spectrometer.

A variety of biochips is available for the capture of biomarkers, inaccordance with the present invention, from commercial sources such asCiphergen Biosystems (Fremont, Calif.), Packard BioScience Company(Meriden, Conn.), Zyomyx (Hayward, Calif.), and Phylos (Lexington,Mass.). Exemplary of these biochips are those described in U.S. Pat.Nos. 6,225,047, 6,329,209, and in PCT Publication Nos. WO 99/51773 andWO 00/56934.

More specifically, biochips produced by Ciphergen Biosystems havesurfaces presented on an aluminum substrate in strip form, to which areattached, at addressable locations, chromatographic or biospecificadsorbents. The surface of the strip is coated with silicon dioxide.

Illustrative of Ciphergen PROTEINCHIP® arrays are biochips H4, SAX-2,WCX-2, and IMAC-3, which include a functionalized, crosslinked polymerin the form of a hydrogel, physically attached. to the surface of thebiochip or covalently attached through a silane to the surface of thebiochip. The H4 biochip has isopropyl functionalities for hydrophobicbinding. The SAX-2 biochip has quaternary ammonium functionalities foranion exchange. The WCX-2 biochip has carboxylate functionalities forcation exchange. The IMAC-3 biochip has nitriloacetic acidfunctionalities that adsorb transition metal ions, such as Cu′ and Ni++,by chelation. These immobilized metal ions, in turn, allow foradsorption of biomarkers by coordinate bonding.

A substrate with an adsorbent is contacted with the urine sample for aperiod of time sufficient to allow biomarker that may be present to bindto the adsorbent. After the incubation period, the substrate is washedto remove unbound material. Any suitable washing solutions can be used;preferably, aqueous solutions are employed. An energy absorbing moleculethen is applied to the substrate with the bound biomarkers. As noted, anenergy absorbing molecule is a molecule that absorbs energy from anenergy source in a gas phase ion spectrometer, thereby assisting indesorption of biomarkers from the substrate. Exemplary energy absorbingmolecules include, as noted above, cinnamic acid derivatives, sinapinicacid and dihydroxybenzoic acid. Preferably sinapinic acid is used.

Once captured on a substrate, e.g., biochip or antibody, any suitablemethod can be used to measure one or more biomarkers in a sample. Forexample, biomarkers can be detected and/or measured by a variety ofdetection methods including for example, gas phase ion spectrometrymethods, optical methods, electrochemical methods, atomic forcemicroscopy and radio frequency methods. Using these methods, one or morebiomarkers can be detected.

In one embodiment, methods of detection and/or measurement of thebiomarkers use mass spectrometry and, in particular, SELDI. SELDI refersto a method of desorption/ionization gas phase ion spectrometry (e.g.,mass spectrometry) in which the analyte is captured on the surface of aSELDI probe that engages the probe interface. In “SELDI MS,” the gasphase ion spectrometer is a mass spectrometer. SELDI technology isdescribed in more detail above.

In another embodiment, an immunoassay can be used to detect and analyzebiomarkers in a sample. An immunoassay is an assay that uses an antibodyto specifically bind an antigen (e.g., a biomarker). An immunoassay ischaracterized by the use of specific binding properties of a particularantibody to isolate, target, and/or quantify the antigen. Thus, underdesignated immunoassay conditions, the specified antibodies bind to aparticular protein at least two times the background and do notsubstantially bind in a significant amount to other proteins present inthe sample. Specific binding to an antibody under such conditions mayrequire an antibody that is selected for its specificity for aparticular protein. For example, polyclonal antibodies raised to abiomarker from specific species such as rat, mouse, or human can beselected to obtain only those polyclonal antibodies that arespecifically reactive with that biomarker and not with other proteins,except for polymorphic variants and alleles of the biomarker. Thisselection may be achieved by subtracting out antibodies that cross-reactwith the biomarker molecules from other species.

Using purified biomarkers or their nucleic acid sequences, antibodiesthat specifically bind to a biomarker (e.g., SerpinA1 polypeptide oralbumin polypeptide) can be prepared using any suitable methods known inthe art. See, e.g., Coligan, Current Protocols in Immunology (1991);Harlow & Lane, Antibodies: A Laboratory Manual (1988); Goding,Monoclonal antibodies: Principles and Practice (2d ed. 1986); Kohler &Milstein, Nature 256:495-497 (1975); Huse et al., Science 246:1275-1281(1989); Ward et al., Nature 341:544-546 (1989).

Generally, a sample obtained from a subject can be contacted with theantibody that specifically binds the biomarker. Optionally, the antibodycan be fixed to a solid support to facilitate washing and subsequentisolation of the complex, prior to contacting the antibody with asample. Examples of solid supports include glass or plastic in the formof, e.g., a microtiter plate, a stick, a bead, or a microbead.Antibodies can also be attached to a probe substrate or a protein chip.

After incubating the sample with antibodies, the mixture is washed andthe antibody-marker complex formed can be detected. This can beaccomplished by incubating the washed mixture with a detection reagent.This detection reagent may be, e.g., a second antibody which is labeledwith a detectable label. Exemplary detectable labels include magneticbeads, fluorescent dyes, radiolabels, enzymes (e.g., horse radishperoxide, alkaline phosphatase and others commonly used in an ELISA),and colorimetric labels such as colloidal gold or colored glass orplastic beads. Alternatively, the biomarker in the sample can bedetected using an indirect assay, wherein, for example, a second,labeled antibody is used to detect bound biomarker-specific antibody,and/or in a competition or inhibition assay wherein, for example, amonoclonal antibody which binds to a distinct epitope of the biomarkeris incubated simultaneously with the mixture.

Methods for measuring the amount or presence of an antibody-markercomplex include, for example, detection of fluorescence, luminescence,chemiluminescence, absorbance, reflectance, transmittance, birefringenceor refractive index (e.g., surface plasmon resonance, ellipsometry, aresonant mirror method, a gating coupler waveguide method orinterferometry). Optical methods include microscopy (both confocal andnon-confocal), imaging methods and non-imaging methods. Electrochemicalmethods include voltametry and amperometry methods. Radio frequencymethods include multipolar resonance spectroscopy. Useful assays arewell known in the art, including, for example, an enzyme immune assay(EIA) such as enzyme-linked immunosorbent assay (ELISA), a radioimmuneassay (RIA), a Western blot assay, or a slot blot assay.

Immunoassays can be used to determine presence or absence of a biomarkerin a sample as well as the quantity of a biomarker in a sample. Theamount of an antibody-marker complex can be determined by comparing to astandard. A standard can be, e.g., a known compound or another proteinknown to be present in a sample. It is understood that the test amountof biomarker need not be measured in absolute units, as long as the unitof measurement can be compared to a control.

When the sample is measured and data is generated, e.g., by massspectrometry, the data may then be analyzed by a computer softwareprogram. In certain cases, a biomarker bound to the substrate can bedetected in a gas phase ion spectrometer. The biomarkers are ionized byan ionization source such as a laser, the generated ions are collectedby an ion optic assembly, and then a mass analyzer disperses andanalyzes the passing ions. The detector then translates information ofthe detected ions into mass-to-charge ratios. Detection of a biomarkertypically will involve detection of signal intensity. Thus, both thequantity and mass of the biomarker can be determined.

Generally, data generated by desorption and detection of biomarkers canbe analyzed with the use of a programmable digital computer. Thecomputer program analyzes the data to indicate the number of biomarkersdetected, and optionally the strength of the signal and the determinedmolecular mass for each biomarker detected. Data analysis can includesteps of determining signal strength of a biomarker and removing datadeviating from a predetermined statistical distribution. For example,the observed peaks can be normalized, by calculating the height of eachpeak relative to some reference. The reference can be background noisegenerated by the instrument and chemicals such as the energy absorbingmolecule which is set as zero in the scale.

A computer can transform the resulting data into various formats fordisplay. The standard spectrum can be displayed, but in one usefulformat only the peak height and mass information are retained from thespectrum view, yielding a cleaner image and enabling biomarkers withnearly identical molecular weights to be more easily seen, in anotheruseful format, two or more spectra are compared, convenientlyhighlighting unique biomarkers and biomarkers that are up- ordownregulated between samples. Using any of these formats, one canreadily determine whether a particular biomarker is present in a sample.

Software used to analyze the data can include code that applies analgorithm to the analysis of the signal to determine whether the signalrepresents a peak in a signal that corresponds to a biomarker accordingto the present invention. Software also can subject the data regardingobserved biomarker peaks to classification tree or artificial neuralnetwork (ANN) analysis, to determine whether a biomarker peak orcombination of biomarker peaks is present that indicates a diagnosis ofintra-amniotic inflammation.

7. Multiple/Combined Analysis of PE

In certain embodiments, methods are provided that are diagnostic and/orpredictive for PE comprising utilizing one or more assays to measure oneor more biomarkers for PE, thereby confirming with the subsequent assaythe results obtained from the previous assays and/or obtainingadditional quantitative and/or qualitative results that improvepredictive value of the test and/or lead to a higher degree ofconfidence in diagnostic tests. For example, initially simple Congo RedRetention assays may be performed (e.g. dot blots) to give firstindication that a subject has developed or is at risk of developingpreeclampsia. This first test may be carried out by the subject withoutthe need for supervision and/or laboratory equipments, or with onlylimited technical expertise and/or equipment required.

If the Congo Red Retention assays is positive, it may be quantified asdescribed herein. This indication may then also be predictive of theseverity of PE. In addition to the Congo Red Retention assays, or asimilar dye-based assay, samples may be subjected to antibody-basedassays described herein, using e.g., antibodies that detect specificconformations of misfolded protein aggregates. These test may be carriedout on the same sample that was used for the Congo Red Retention assayor on a second sample from the same subject and these tests may becarried out after Congo Red Retention assay or at substantially the sametime. The assays using antibodies that detect specific conformations ofmisfolded protein aggregates may, in certain embodiments, be used toconfirm the result obtained using the Congo Red Retention assay or maybe used to obtain additional qualitative information, e.g., correlatingthe presence or absence of specific oligomeric protein conformationswith the likelihood to develop PE or as an indication of the severity ofPE. In addition to the assays that are based on protein aggregates foundin PE samples (i.e., congophilia/Congo Red Retention and antibody-basedprotein aggregate analysis) additional test may be conducted to eitherconfirm the results obtained in the previous assays or to obtainadditional quantitative or qualitative data. For example, additionalassays may be performed measuring the absence or presence and/or therelative amount of one or more biomarkers described herein, includingassays to detect the PE biomarkers albumin, SerpinA1, sFlt-1, P1GF,VEGF, using assays such as ELISA or e.g., analyzing protein fragments bySELDI-TOF, etc. Additionally, predictive ratios may be calculated forthe biomarkers, such as e.g., sFlt/P1GF [uFP]. Further, the presence orabsence and/or relative level of additional proteins may be used fordiagnosis of PE, such as immunoglobulins, ceruloplasmin and insulininducible protein 6-16 in protein aggregates of urine.

It should be appreciated that the tests and assays described herein maybe carried out in any order and may be conducted alone or in anycombination and the invention is not limited in this aspect. It haspreviously been shown by Applicant and described herein that any one ofthe tests and assays described is sufficient to detect PE and to aiddiagnosis and prognosis.

8. Treatments

Protein conformational disorders such as Alzheimer's, light chainamyloidosis and prion diseases are propagated by amyloid fibrilformation and aggregation due to defective folding of cellular proteinsinto aberrant 3D structures. It has recently been observed that solublepre-amyloid oligomers (intermediates in fibril assembly) haveproteotoxic effects leading to endothelial damage and oxidative stress.Applicants have previously shown that endothelial damage and oxidativestress play pathogenic roles in severe preeclampsia (sPE). Applicantshave now unexpectedly found that PE is a conformational disorderassociated with protein misfolding and aggregation, characterized byamyloid-like assembly of proteins. Applicants further established byantibody staining with conformation-specific polyclonal and/ormonoclonal antibodies that the misfolded intermediates found in theurine of subjects with PE have a propensity to assemble into pore-likestructures (amyloid channels) that may play a role in clinical diseasemanifestations, such as e.g., endothelial damage and oxidative stress.Applicants have reasoned that the accumulation of abnormal proteinaggregates in the placenta of patients diagnosed with preeclampsiaindicates that these protein aggregates are a causative factor in thepathology of this disease. In certain embodiments, therapeuticalinterventions for preeclampsia, utilizing immunological orpharmacological strategies, are provided based on blocking the formationof misfolded protein oligomers that may assemble into amyloid channels.In certain embodiments, treatments for PE are provided. In certainembodiments, treatments are based on therapeutic regimens that werefound to reduce severity of other misfolding disorders such as forexample Alzheimer's disease. Research into novel therapeutic strategiesfor the treatment of similar diseases of abnormal protein aggregation,such as Alzheimer's disease and other amyloidoses, has identified agentsthat can inhibit the development of new protein aggregates and/ordecrease the burden of pre-existing protein aggregates. The finding thatPE is associated with abnormal protein aggregates presents avenues fortreatment.

Multiple approaches and agents, e.g., small molecular weight inhibitors,peptidic inhibitors of fibril and/or oligomer formation, vaccinationagainst one or more of the components of the protein aggregate, passiveimmunization with antibodies or antibody fragments against one or moreof the components of the protein aggregate, among other approaches havebeen developed for the inhibition and/or reversal of beta-amyloidaggregation in the treatment of Alzheimer's disease. Some of theseagents have been shown to inhibit the aggregation of proteins other thanbeta-amyloid. For example, p-Aminophenol and 2-Amino-4-Chlorophenol andderivatives (Cell Biochemistry and Biophysics 44: 549-553 (2006)) havebeen shown to inhibit aggregation as well.

In certain embodiments, methods of treating preeclampsia in patientsusing protein aggregation inhibitors to inhibit the de novo formation ofprotein aggregates and/or reverse the formation of preexistingaggregates are provided. In certain embodiments, agents that stabilizethe native conformation of aggregate-prone proteins are provided thatdecrease the rate of abnormal folding and consequent aggregation. Incertain embodiments, small molecular weight inhibitors, peptidicinhibitors of fibril and/or oligomer formation, vaccination against oneor more of the components of the protein aggregate, passive immunizationwith antibodies or antibody fragments against one or more of thecomponents of the protein aggregate are provided for the treatmentand/or prevention of preeclampsia. In certain embodiments, anti-betaamyloid agents (e.g., those summarized in Drug Discovery Today:TherapeuticStrategies 1: 7-12 (2004) and references contained thereinare provided for the treatment and/or prevention of preeclampsia.

Applicants have also found that SerpinA1 (alpha-1 antitrypsin) and/orpeptide fragments of SerpinA1 is a component of the of the proteinaggregates found in preeclampsia. It has previously been shown thatSerpinA1 can serve as a biomarker for early detection of preeclampsia(U.S. application Ser. No. 12/084,004 (PCT/US2006/042585), incorporatedherein by reference in their entirety).

As used herein the term “SerpinA1 polypeptide” refers to full-lengthSerpinA1 polypeptide and also to a polypeptide that is a fragment offull-length SerpinA1 polypeptide. SerpinA1 has been previouslyidentified as a serine protease inhibitor, and is also known as alpha 1antitrypsin. SerpinA1 polypeptide has Genbank Accession No. P01009. Itwill be understood that SerpinA1 polypeptides encoded by alternativealleles of SerpinA1 may also be used to detect the presence ofpreeclampsia in subjects. For example, SerpinA1 polypeptides encoded byM1A, M2, and/or M3 alleles of SerpinA1 may be used in methods of theinvention to diagnose and/or assess preeclampsia in subjects. SerpinA1polypeptides are synthesized in the liver and trophoblast and arepresent in multiple forms that are unrelated to SerpinA1 'santiproteolytic activity. SerpinA1 polypeptide is highly susceptible tooxidation and intensive oxidative stress induces SerpinA1 oxidation. Apolypeptide that is a fragment at the C-terminus of full-length SerpinA1polypeptide induces oxidative burst and neutrophil chemotaxis in vitro.

The identification of SerpinA1 in urinary samples of subjects with PEindicates that preeclampsia has a similar disease etiology to otherdisorders such as alpha-1 antitrypsin deficiency which, due to theaccumulation of misfolded alpha-1 antitrypsin leads to damage ofhepatocytes and cirrhosis of the liver (N. Engl. J. Med. 346: 45-53(2002); J. Clin. Inv. 110: 1585-1590 (2002)). In certain embodiments,therapeutic agents that prevent the formation of abnormal aggregates ofSerpinA1 protein or its fragments are provided. In other embodiments,agents are provided that can dissociate existing SerpinA1 aggregates orits fragments. For example, on such agent is Trimethylamine N-oxide andrelated compounds (Am. J. Respir. Cell Mol. Biol. 24:727-732 (2001)).Another example is the FLEAIG peptide (SEQ ID NO: 7) and relatedpeptides and derivatives (Am. J. Respir. Cell Mol. Biol. 35: 540-548(2006)). In certain embodiments, Trimethylamine N-oxide and relatedcompounds or FLEAIG peptide (SEQ ID NO: 7) and related peptides andderivatives are provided as useful therapeutic agents for the treatmentand/or prevention of preeclampsia.

As will be appreciated by those of ordinary skill in the art, evaluationof a treatment also may be based upon an evaluation of the symptoms orclinical end-points of preeclampsia. Thus, methods of the invention areuseful for determining the onset, progression or regression of acondition that is characterized by one or more of the biomarkers (suchas e.g., SerpinA1, albumin, sFlt-1, P1GF, VEGF, misfolded proteinaggregates/congophilia) described herein in a pregnant subject. In someinstances, methods of the invention can be used to detect levels of oneor more of the biomarkers described herein in subjects diagnosed ashaving preeclampsia. In other instances, methods of the invention can beused to obtain measurements that represent the diagnosis of preeclampsiain a subject. In some instances, a subject may be already be undergoingdrug therapy for preeclampsia, while in other instances a subject may bewithout present preeclampsia therapy.

The type of treatment for preeclampsia selected may be based, in part,upon selecting pregnant women who have abnormally high levels of one ormore of the biomarkers described herein (such as SerpinA1, albumin,sFlt-1, P1GF, VEGF) and/or are shown to have misfolded proteinaggregates associated with preeclampsia and exhibit congophilia.Treatments may include administration of a particular type of drug, anactivity change, or a dietary change, which may be based at least inpart on the presence or absence of an indication of preeclampsia (e.g.,detection of one or more of the biomarkers, congophilia/misfoldedprotein aggregates and/or specific protein oligomer conformations,described herein). Such subjects may already be receiving a drug fortreating preeclampsia. It may be appropriate according to the inventionto alter a therapeutic regimen for a subject, based upon the measurementof the level of one or more of the biomarkers, congophilia/misfoldedprotein aggregates and/or specific protein oligomer conformationsdescribed herein using a method set forth herein. This can be understoodin connection with treatment of preeclampsia. A subject may be free ofany present treatment for preeclampsia and monitoring of one or more ofthe biomarkers, congophilia/misfolded protein aggregates and/or specificprotein oligomer conformations described herein may allow selection ofthe most efficacious treatment regimen.

9. Kits

In some embodiments, the instant invention provides kits useful in themethods of the invention. Reagents may be labeled compounds or agentscapable of detecting congophilia or misfolded protein aggregates in aurine sample. In certain embodiments, the kits may comprise a surfacethat has affinity to proteins that can be contacted with the sample(e.g., a urine sample). The kit may further comprise a dye (e.g., CongoRed) that associates with misfolded protein aggregates associated withpreeclampsia and can be contacted with the sample. The kit may alsocomprise a vessel, tube or container suitable for mixing the sample withthe dye, or incubating the surface that has affinity to proteinscomprising the bound proteins of the sample. The kit may contain one ormore washing solutions suitable to remove unincorporated dye. The kitmay further comprise positive and negative controls to allow for controlof sufficient incorporation and/or washing, e.g., provided on a “teststrip” comprising the surface that has affinity to proteins.

In some embodiments, the instant invention provides kits useful in themethods of the invention. Reagents may be labeled compounds or agentscapable of detecting, in a urine sample, the presence of misfoldedprotein aggregates and means for determining the amount of thepolypeptide (e.g., an antibody that binds the polypeptide). Suitablereagents for binding with a polypeptide corresponding to a biomarkeruseful in a method of the subject invention include antibodies, antibodyderivatives, antibody fragments, and the like. For antibody-based kits,the kit can comprise, for example: (1) a first antibody (e.g., attachedto a solid support) which binds to a polypeptide corresponding to abiomarker of the invention; and, optionally, (2) a second, differentantibody that binds to either the polypeptide or the first antibody andis conjugated to a detectable label.

Suitable PE-biomarkers that may be detected using such kits includesFlt-1, P1GF, VEGF, albumin, SerpinA1, IFI6 and ceruloplasmin. Incertain embodiments, kits comprise antibodies specific for certainconformations of misfolded proteins, e.g., fibrillar conformations andthe like. The antibody may bind to specific protein aggregateconformations from the sample tested/analyzed by the kit. In certainembodiments, wherein the sample is a urinary sample comprisingPE-associated protein aggregates which comprise immunoglobulins, thesecondary antibody is preadsorbed with Ig to reduce or preventnon-specific binding of the secondary antibody to the protein aggregatecomprising immunoglobulins.

The kits, in certain embodiments, can also comprise other components,such as a buffering agent, a preservative, or a protein stabilizingagent. The kit can further comprise components necessary for detectingthe detectable label (e.g., an enzyme or a substrate).

Each component of the kit can be enclosed within an individual containerand all of the various containers can be within a single package, alongwith instructions for interpreting the results of the assays performedusing the kit. The container may be pretreated with a stabilizing agentand/or a stabilizing agent may be a component of the kit.

EXAMPLES Example 1 Methods Protocol Congo Red Dot Blotting of UrineSamples (Red Dot Test):

1. Centrifuge obtained urine samples at 1500×g, 15 minutes and 4° C.2. Measure urine protein in the spun urine using the Pierce BCA kit(Thermo Scientific Cat#23225). Urine samples may require a 12-folddilution for protein measurement. Urine samples from women that arepotentially preeclamptic have wide variations in protein concentration.Protein levels in urine also vary widely in healthy people depending onthe state of hydration. For this reason it is preferred to equalize thetotal protein concentration. Out of over 680 urine samples obtained fromwomen only approximately 5% (37/681) of urine samples measured<2 mg/mLtotal protein. Approximately 60% of urine samples had over 6.6 mg/mLtotal protein. Generally, in most of the cases there is no need to drythe samples, e.g. in the SPEEDVAC®. The drying method was validated andit was found that sample drying does not change the Red Dot Test result.For consistency, SPEEDVAC® drying was used for all assays.3. Add the volume of sample that contains 200 μg protein to a conicalcentrifuge tube and concentrate to dryness using, e.g., a SPEEDVAC®.4. Prepare the Congo Red stock solution (5 mg/ml-) Congo red (Sigma,Cat#C6277) in water. Centrifuge at 14,000×g, 10 minutes to spin downundissolved powder. The stock solution should be freshly made, andshould be used (i.e. added to the urine samples) within approximately 1hour of preparation. One may choose to prepare a Congo Red workingsolution (0.1 mg/ml) by diluting 50-fold the stock solution.5. Make Blank sample by adding 2 μl Congo red stock to 0.1 mL PBS(phosphate buffer).6. Remove dried urine samples from SPEEDVAC® and add 30 μL of Congo Redworking solution and vortex well.7. Incubate 1 hour with vigorous shaking (on a vortex) to achieve Congored binding.8. After 1 hour, apply duplicate dots of 5 μl sample with Congo red onnitrocellulose membrane (0.2 micron mesh) in duplicate. Each dot shouldpreferably contain 33.3 μg protein. Also apply the Blank sample induplicate. If more than one sheet is necessary each sheet should containdots from the Blank sample. Preferably a positive control sample isincorporated in each run. One may choose to make an application gridwhich can be placed on a transiluminator and on top of it thenitrocellulose membrane can be placed so that the dots can be spaced inan orderly fashion and so that it is possible to track exactly whereeach sample has been placed.9. Let membrane dry for 15 minutes.10. Wash in water for 3 minutes.11. Photograph (all spots should look similar in staining intensity atthis point).12. Wash in 50% methanol for 3 minutes then in 70% methanol for 1minutes and then in 90% methanol until the red in the Blank sampledisappears (generally 7-10 minutes).13. Photograph (spot intensity, e.g. positive control sample: Blanksample) should look different).14. Scan the images and calculate the optical density (OD) of each dotbefore and after the methanol wash using any densitometry software, e.g.NIH Image J.15. Calculate the % Congo Red Incorporation (CRI) for all samples as(Average OD of spots from same sample before methanol wash/Average OD ofBlank from the same nitrocellulose sheet)×100.16. Calculate the % Congo Red Retention (CRR) for all samples includingthe Blank as (Average OD of spots from same sample after methanolwash/Average of spots from same sample before methanol)×100; thensubtract CRR of the Blank sample from all values of the dots from urinesamples. If the methanol washing was complete there should not be anyresidual red visible in the dots from the Blank and their OD averageshould be 0.17. Call samples with CRR>20% Red Dot Test positive and those CRR<15%Red Dot Test negative.18. Repeat test if CRR is between 15-20%. It has been found that thediagnostic and prognostic cut-off of the 223 samples analyzed by ususing the Red Dot Test is approximately 16.1%.

It should be appreciated that all incubation and preparation (e.g.,centrifugation) times, volumes and concentrations are the preferredtimes, volumes and concentrations. However, other incubation andpreparation times, volumes and concentrations may also lead to reliabletest results and can be established by one of skill in the art withoutundue experimentation, and the incubation and preparation times, volumesand concentrations described here are non-limiting. Further, it shouldbe appreciated that the chemicals and equipment described herein arenon-limiting and other equivalent chemicals and/or equipment may beused. Further, it should be appreciated that some steps and the order ofthe steps described herein may be optional and some steps and the orderof the steps may be changed, omitted, or combined. Such changes andmodifications would be apparent to one of ordinary skill. For example,incubation times of less than 1 hour (step #7), adding a non-denaturingdetergent (e.g. Tween 20) to the urine sample, or substituting methanolwith ethanol or isopropanol in the washing steps will also result in areliable test.

Study Design:

110 women were enrolled prospectively in 3 groups: normotensive controls(CRL n=49, GA: 28 [21-34] weeks); chronic hypertension (cHTN n=12, GA:29 [24-34] weeks) and severe PE (sPE n=49, GA: 30 [24-34] weeks). Inaddition, 34 asymptomatic pregnant women at high-risk of PE werefollowed longitudinally. Urine congophilia was quantified by dot blotfixation and spectral shift assays on equal amounts of protein.Congophilic proteins were identified by tandem mass spectrometry andvalidated by Western blot. A urinary proteomic fingerprint was generatedusing SELDI-TOF. Placental congophilia was examined by polarizedmicroscopy.

Results

FIG. 1 shows a photograph of a nitrocellulose membrane before (left) andafter wash (right) following Congo Red Dot Test of urine samples (5μL/spot were applied to nitrocellulose). As can be seen from the Figure,Congo Red stains the samples similarly well before the wash, but isspecific for urine samples obtained from women with severe preeclampsia(sPE) after the wash (with the blank being negative). FIG. 2 shows aphotograph of a nitrocellulose membrane before and after wash followingCongo Red Dot Test of urine samples (33 μg/spot were applied tonitrocellulose) of a cross-sectional cohort (24 samples). Samples withboxed number were from women with manifest severe preeclampsia (sPE). Ascan be seen from the Figure, Congo Red stains the samples similarly wellbefore the wash, but is specific for urine samples obtained from womenwith severe preeclampsia after the wash. FIG. 3 shows a photograph of anitrocellulose membrane before and after wash following Congo Red DotTest of urine samples (33 μg/spot were applied to nitrocellulose) of alongitudinal cohort (28 samples). Six pregnant women were followed withrepeat analysis of urine throughout pregnancy. Patients #2 and #5developed preeclampsia. The boxed samples obtained from patient #5 werefrom the time of clinically manifest disease. The boxes corresponding tosamples U348i and U348j for patient #5 were postpartum after medicallyindicated delivered for preeclampsia (emergency C-section). As shownhere, both patients (#2 and #5) had abnormal Congo red dot urine test(red dot positive) long before they were diagnosed and admitted forpreeclampsia. FIG. 14 shows a graph for the prediction of indicateddelivery for preeclampsia in a longitudinal cohort. 36 women athigh-risk (n=30) and low-risk (n=6) were studied using the Congo RedTest. Four women in this cohort developed severe preeclampsia and had anindicated preterm delivery. The data shows that women that laterdeveloped PE had high urine congophilia in the first sample that wastested. It was not possible to obtain earlier sample to test if thesewomen had a positive test also before pregnancy. FIG. 4 shows a bargraph of Congo Red Retention (CRR) normalized for amount of protein asdescribed in women with severe preeclampsia (sPE), chronic hypertension(cHTN) and normal pregnant controls (CRL). As shown, CRR is clearlyassociated with and indicative of severe preeclampsia (sPE). The urinewas tested at the time of admission with diagnosis of severepreeclampsia. FIG. 5 shows ROC curves of Congo Red Retention (CRR) andCongo Red incorporation (CRI) coefficients to (A) diagnose, and (B)predict a mandated delivery for preeclampsia (223 urine samples from 114different pregnant women). Some women had sequential urine samplesanalyzed throughout pregnancy. FIG. 6 shows that Congo Red Retention(CRR) of urine proteins is significantly correlated with the presenceand severity of preeclampsia as determined by the abnormal proteomicprofile (UPSr).

FIG. 7 shows that Congo Red Retention (CRR) of urine proteins issignificantly correlated with the ratio indicator uFP: log[sFlt-1/PIGF×100], as described herein and also in (U.S. Publ. No:US-2006-0183175; PCT/US2005/047010). FIG. 8 shows that similar withmisfolded proteins of other diseases and indications (e.g., Alzheimer'sand prion disease), misfolded urine proteins in preeclampsia can beseparated by methods such as (A) Congo red affinity by dot blot (boxedsamples are from women with clinically manifest severe preeclampsia(sPE)) as described herein, (B) gel filtration, or (C) centrifugationand washing of the precipitated Congo red bound proteins with water(e.g. for (B) and (C)).

In further experiments, placental sections were stained with Congo redand showed cloud-like birefringent material deposited in PE but not CRLsyncytiotrophoblasts. FIG. 15 A shows placental sections from threewomen, two with preterm delivery for severe preeclampsia (A-F) andanother with idiopathic preterm birth (Control, G-H) that were stainedwith Congo Red and examined microscopically in either white light (A, D,G) or polarized light (B, C, E, F, H and I). Panels C and F are highermagnifications (640×) of the squared areas in Panels B & E, respectivelyAs seen in B and E, the placenta of the preeclamptic woman showscloud-like grey-blue (B & C) or grey-green (E & F) birefringent materialdeposited in the syncytiotrophoblast layer (red arrows). Panel I:polarized light image of a brain section from a patient with Alzheimer'sdisease that has been stained with Congo Red in the same conditions.

FIG. 15 B shows imaging of Congo Red positive precipitated material inpreeclamptic urine. After Congo Red binding and centrifugation theprecipitated material (A) is washed with water 3 times (repeating thecentrifugation between the washes), resuspended in a drop of water andeither placed on a microscope slide for imaging in polarized light (B)or on a grid for electron microscopy after staining positively (C) ornegatively (D) with 1% uranyl acetate. The arrows point to anon-cellular structure, likely a fibril.

In further experiments, Congophilic material as well as the bandsimmunoreactive for conformational antibodies were run on SDS PAGE andsubjected to mass spectrometry. Fragments of antitrypsin, ceruloplasmin,heavy- and light-chain IgG were identified in congophilic proteinuria ofPE and in the bands immunoreactive for conformational antibodies.Western blot for SERPINA1 in congophilic material was performed and manyfragments in a ladder pattern that reacted with the antibody wereobserved. The presence of ceruloplasmin and IFI6-16 in PE urine wasconfirmed by western blot and/or dot blot. Light chains were confirmedby ELISA. FIG. 16 shows urine samples from two women with severepreeclampsia (U647 and U648) that were loaded on duplicate 4-20%reducing SDS PAGE gels without (lanes 1-2 and 5-6) or with enrichment ofmisfolded proteins by Congo Red affinity precipitation (lanes 3-4 and7-8, red asterisk). The left panel shows the gel after staining of totalproteins with Coomassie blue stain (Lanes 1-4). Many new bands becamevisible in the samples precipitated with Congo Red. The right panelshows the immunoreactivity for SerpinA1 of proteins transferred tonitrocellulose. The additional fragmentation (ladder) pattern is visiblein lanes 7 & 8 but not in 5 and 6. Molecular weight markers (MW) areshown on each panel.

This also implies that Congo Red can be efficiently used to enrichbiological samples (in this case preeclamptic urine) in misfolded andaggregated proteins which can be further studied.

In summary, the data show that PE is characterized by a marked increasein urine congophilic proteins compared to CRL and cHTN, independent ofGA (P<0.001). In women followed longitudinally who developed sPErequiring early delivery (n=4), congophilic urine proteins appeared 8-10weeks prior to clinical manifestations. Congo red binding to PE urineproteins resulted in a red-shift in absorbance similarly seen for otheramyloid supramolecular protein structures. Urinary congophiliacorrelated with the proteomic profile characteristic of PE (r=0.89,P<0.001).

The data provide evidence that PE is a pregnancy-specific diseasecharacterized by supramolecular amyloid-like assembly of proteins andcongophilic proteinuria. Detection of urine congophilia by dot blotfixation and/or spectral shift assays are indicative of a patient withpreeclampsia and provide a method for diagnosis of preeclampsia. Urinarycongophilic protein aggregates, detected as described, are not onlydiagnostic of an existing preeclampsia, but are also predictive of thefuture development of preeclampsia (clinical outcome). In addition todetecting the presence of congophilic protein aggregates in the urine,placental tissue samples stained with Congo Red display characteristicfeatures of such protein aggregates. This methodology of detecting CongoRed staining in the placenta also has application in the diagnosis ofpreeclampsia.

It should be appreciated that in addition to Congo Red the presence ofprotein aggregates may also be detected by a variety of other agents andmethodologies (e.g., Thioflavin S, amongst others) that are known in theart.

Example 2 Methods Study Design:

347 pregnant women were enrolled prospectively in the following groups:normotensive controls (CRL n=98, GA:27[7-42 weeks]); chronichypertension (cHTN n=40, GA:32[11-41 weeks]), gestational hypertension(gHTN n=8 GA:37[26-39 weeks]), mild PE (mPE n=36, GA:36 [24-41 weeks]),severe PE (sPE n=117, GA:32[22-42 weeks]), superimposed PE (spPE n=33GA:33[18-40 weeks]). 35 asymptomatic women were tested seriallythroughout gestation. A “Congo Red (CR) Dot” test was standardized withequal urine protein and objectively quantified within minutes as % CongoRed retention (CRR). CRR was evaluated for its ability to predict anindicated delivery for PE (IND) compared to protein-to-creatinine ratio(P/C) and the previously validated urine sFlt1/P1GF ratio, as describedherein and also in (U.S. Publ. No: US-2006-0183175; PCT/US2005/047010).

Results:

PE is characterized by increased excretion of misfolded proteins withaffinity for the azo dye Congo Red (CR), also used to detect aberrantamyloidal aggregates in Alzheimer's and prion disease. A “Congo Red (CR)Dot” test CRR was designed and validated as a diagnostic and prognostictest for PE based on urine congophilia as a measure of global proteinmisfolding load in pregnancy. FIG. 9A shows % Congo Red Retention (CRR)of urine proteins of different treatment groups (CRL: control; cHTN:chronic hypertension; gHTN: gestational hypertension; mPE: mildpreeclampsia; sPE: severe preeclampsia; spPE: superimposedpreeclampsia). 61% (211/347) of women had IND: CRL: 4%; crHTN: 40%;gHTN: 75%; mPE: 69%; sPE: 99%; spPE: 100%. 77% (162/211) of INDsoccurred preterm and 51% (107/211)<34 weeks GA. CRR was elevated in mPEand further increased in sPE and spPE independent of GA. Women requiringIND had elevated CRR at enrollment (FIG. 9B, P<0.001). FIG. 9C showsthat among women followed longitudinally, 11% (4/35) had preterm IND. Inthis group, CRRs was increased 14±4 weeks prior to clinically manifestPE. CRR had higher accuracy in predicting IND compared to urine ratio ofsFltl/P1GF (P=0.014) and urine protein/creatine ratio (P/C) (P<0.001)(FIG. 9 C).

In summary, assessment of global protein misfolding load by CRR is asimple diagnostic test for PE and for prediction of IND, an importantcontributor to preterm birth.

Example 3 Methods Study Design:

111 urine samples from women enrolled in 3 groups: sPE (n=49, GA: 28±1weeks), chronic hypertension (cHTN n=12, GA: 29±1 weeks) andnormotensive controls (CRL n=50, GA: 28±1 weeks) were analyzed. Equalamounts of urine protein was subjected to dot blot using 3conformation-specific antibodies recognizing prefibrillar solubleoligomers (A11, Invitrogen), ring-shaped protofibrils (Officer) orfibrils (OC). Specificity was confirmed by omitting the primaryantibodies. Identity of aggregated component proteins was sought by massspectrometry and validated by Western blot with sequence-specificantibodies.

Results

Protein conformational disorders such as Alzheimer's, light chainamyloidosis and prion diseases are propagated by amyloid fibrilformation and aggregation due to defective folding of cellular proteinsinto aberrant 3D structures. Soluble pre-amyloid oligomers(intermediates in fibril assembly) have proteotoxic effects leading toendothelial damage and oxidative stress.

Some important features in the pathogenesis of preeclampsia are vascularendothelial activation followed by vasospasm. Theories of its causeinclude abnormal implantation and development of the placenta, oxidativestress, impaired endothelial prostanoid and nitric oxide homeostasis,genetic polymorphisms, abnormal circulating autoantibodies and anabnormal maternal systematic inflammatory response (Buhimschi I A et al.Hum Reprod Update 1998; 4:25-42; Ward K et al; Nat Genet 1993; 4:59;Wallukat G et al. J Clin Invest 1999; 103:945-952; Fass M M et al. Am JObstet Gynecol 1994; 171: 158-64; Roberts J M et al. Lancet 1993; 341:1447-51). As endothelial damage and oxidative stress play pathogenicroles in severe preeclampsia (sPE) the nature of urinary solublepre-amyloid oligomers associated with PE was identified andcharacterized.

Antibodies that were used to detect urinary soluble pre-amyloidoligomers included those that detect proteins in (i) a prefibrillarsoluble oligomer conformation, such as the “A11” antibody amongstothers, (ii) a ring-shaped protofibril conformation, such as the“Officer” antibody amongst others, and (iii) a fibril conformation, suchas the “OC” antibody amongst others.

The urine dot blots comprising urinary protein aggregates presented aproblem of nonspecific binding of the A11 antibody. It was laterestablished (by mass spectrometry of the urinary protein samples) thatthe urinary protein aggregates of the samples also comprise misfoldedhuman immunoglobulin (IgG) heavy- and light chains that were recognizednon-specifically by (i.e., cross-reacted with) most of the secondaryantibodies used. It was challenging to find a procedure which minimizedthis cross-reactivity. After screening numerous secondary antibodies forspecificity it was found that it was critical for specificity to usesecondary antibodies that were preadsorbed with human IgG.

The sensitivity of assays using the A11 polyclonal antibody to detecturinary protein aggregates was markedly improved if the secondaryantibody was preadsorbed with human IgG to reduce non-specific bindingof the secondary antibody to the urinary protein aggregates comprisingmisfolded human immunoglobulin (IgG) heavy and light chains. Using thisassay methodology, positivity in the A11 assay correlated with severityof the symptoms. FIG. 10 shows a photograph of a western blot of anitrocellulose membrane with spotted urine samples probed with threepolyclonal antibodies A11, OC, and Officer. The 3 polyclonal antibodies(A11, specific for a wide range of conformations), OC (specific forfibrils) and Officer (specific for some annular conformations) showedimmunoreactivity to samples from preeclampsia patients (as indicated:urine, blood, cerebrospinal fluid (CSF) and placenta lysates; PE:preeclampsia; CRL: control). The Officer antibody is thought to bespecific for protein conformations that might form channels or pores inRBC analogous with amyloid channels and may explain hemolysis inpatients with preeclampsia. Urine, blood, cerebrospinal fluid andplacenta lysates were also tested. The data show that PE is aconformational disorder characterized by amyloid-like assembly ofproteins. Concurrent A11 and Officer staining suggests that themisfolded intermediates have a propensity to assemble into pore-likestructures (amyloid channels) that may play a role in clinical diseasemanifestations.

FIG. 11 shows representative dot blots of urine samples from women inthe 3 categories: preeclampsia (PE), cHTN and CRL. Each spot contained40 mg urine protein. Red arrows mark rows where samples are spotted. Thefilm is compared with the sample application grid and the position ofeach sample circled in black. It was found that many women withpreeclampsia and Congo red positive test show A11 immunoreactivity inurine samples, although not all Congo red positive women have A11positivity. The data indicate that A11 positivity correlates withsymptom severity.

Women with sPE had increased A11 and Officer (sPE: 42±8 vs. cHTN: 9±6vs. CRL: 3±1 U/μg, P<0.001) but not OC urine immunoreactivity,independent of GA. Urine A11 and Officer dot blot staining intensitycorrelated with severity of hypertension (P=0.007) and proteinuria(P=0.005).

In further experiments, it was tested which proteins are involved informing the aggregates recognized by A11 in PE urine. Two high molecularweight bands which were also A11 positive in non-reducing SDS PAGE werecut out. A tryptic digests was performed and the sample was submitted tothe Keck facility for protein identification of the soluble oligomers insPE urine. The following identities were confirmed by mass spectrometry:immunoglobulin heavy and light chains, ceruloplasmin and interferoninducible protein 6-16 protein (GIP3, IFI6-16), which was found tointeract with Alzheimer's presenilin-2 protein to regulate apoptosis.The presence of IFI6-16 in dot blots of preeclamptic urine was confirmedusing an anti IFI6-16 antibody. The antibody was a mouse polyclonal fromNovus Biologicals Inc, Littleton, Colo. Catalog # H00002537-A01.

The finding that proteins in the urine from preeclampsia patients, thatwere immunoreactive with the A11 antibody, include immunoglobulin heavyand light chains, ceruloplasmin and the interferon-inducible protein6-16, provides evidence that quantitation of one or more of theseproteins in protein aggregates in the placenta and/or the urine also hasutility in the diagnosis or prognosis of preeclampsia.

Example 4 Assessment of Samples

Antigen and antibody preparation is described in Kayed et al., “Fibrilspecific, conformation dependent antibodies recognize a generic epitopecommon to amyloid fibrils and fibrillar oligomers that is absent inprefibrillar oligomers,” Molecular Neurodegeneration 2007, 2:18 whichreads as follows:

Fibril antigens were prepared by stirring 2 mg/ml Aβ42 peptide in 50%HFIP/H2O, 0.02% sodium azide for 7 days. Afterwards, the HFIP wasevaporated under a stream of nitrogen and the sample was stirred for anadditional 7 days and dialyzed against PBS (molecular weight cut off10,000 Da). The resulting fibrils were checked by EM and the purity wasconfirmed by the absence of oligomers using anti-oligomer antibody. Theantigens were each used to immunize two New Zealand white rabbits(Pacific Immunology Corp., Ramona, Calif., 92065) according to protocolsapproved by IACUC. Each rabbit immunized with 500 μl of antigen incomplete Freund's adjuvant (CFA), and then boosted twice at four weekintervals with 500 μl of antigen in Incomplete Freund's Adjuvant (IFA).

Fibril and oligomer preparation: Aβ fibrils and fibrillar oligomers wereprepared by dissolving 0.3 mg of lyophilized Aβ42 in 150 μl ofhexafluoro-2-propanol (HFIP) for 10-20 minutes at room temperature. Theresulting Aβ solution was added to DD H₂O in a siliconized Eppendorftube to 80 μM concentration. After 10-20 min incubation at roomtemperature, the samples were centrifuged for 15 min. at 14,000×G andthe supernatant fraction (pH 2.8-3.5) was transferred to a newsiliconized tube and subjected to a gentle stream of N₂ for 10 min toevaporate the HFIP. The sample was then stirred at 500 RPM using aTeflon coated micro stir bar for 24 hours at 22° C. This method wasoriginally reported for preparing A11 positive prefibrillar oligomers,but more recent work indicates that it also produces fibrillar oligomersthat are OC positive (Kayed et al., “Common structure of soluble amyloidoligomers implies common mechanism of pathogenesis,” Science 2003,300(5618):486-489). Fibrils were separated from fibrillar Aβ42 oligomersby centrifuged at 100,000×G for 1 hour at 4° C. The supernatantcontaining fibrillar oligomers and pellet fraction containing fibrilswere separated and collected. The pellets were resuspended in an equalvolume of H2O. Alternatively, 1 mg of lyophilized Aβ42 was dissolved in200 μl of DMSO and incubated at room temperature for 10-15 minutes toform fibrillar oligomers. The fibrillar oligomers in DMSO werefractionated according to size using a TSK-GEL SuperSW2000 column (TosohBioscience LLC) in 10 mM Phosphate, pH 7.4 at a flow rate of 0.3 ml/min.

Results

Because the A11 antibody is non-selective for oligomeric shapes it wasdetermined that further confirmation using monoclonal antibodies wouldprovide additional useful information. Consequently urine samples thatwere either A11 positive or negative were tested using a panel ofmonoclonal antibodies, raised against the same antigen as the A11antibody (Science 300: 486-489, 2003), with preferential affinities fordifferent types of prefibrillar protein oligomer conformations andcompared to the A11 antibody. Ten identical strips containing urinesamples of women with preeclampsia (PE) or controls (CRL) weregenerated. FIG. 12 shows A11 immunoreactivity (strip 1) in thelaboratory at Yale. Strip 2 was blotted using the same reagents andmethods except that the A11 antibody was omitted and served as thecontrol for non-specific binding of the secondary antibodies. Theremaining strips were sent to the laboratory at UC Irvine.

FIG. 13 shows a photograph of a western blot of a nitrocellulosemembrane with spotted urine samples (PE: preeclampsia; CRL: control)probed with monoclonal antibodies (M204, M205, M118, M89, M09, M55) andpolyclonal A11 (A11) and negative control (Neg.) omitting the primaryantibodies to control for non-specific binding of the secondaryantibody. This experiment provided independent confirmation of theimmunoreactivity of the A11 antibody to preeclampsia urine samples anddemonstrated that some more selective monoclonal antibodies also showedimmunoreactivity. These findings show that monoclonal antibodies thatrecognize different conformations of prefibrillar protein oligomers,such as monoclonal #M204, #M205 and #M89 are useful in the diagnosis ofpreeclampsia.

Urine samples from women with preeclampsia (PE), but not control casesstained strongly with monoclonal antibody M204 and weakly withmonoclonal antibody M205. A subset of the preeclampsia cases alsostained with the annular protofibril specific monoclonal M89. Of thesamples tested in FIG. 13, the PE case that stained strongest with M89also was the one that had incipient clinical deterioration consistentwith HELLP (Case #4). Conversely, Case #6 (negative for M89) butpositive for M204, M205 and A11, is of interest because the diagnosis ofsevere preeclampsia was based by the sole criterion of pulmonary edemaunlike the other PE women who were classified as having severepreeclampsia by blood pressure and/or proteinuria alone. Case #6subsequently developed peripartum cardiomyopathy which requiredextensive circulatory support in intensive care unit. The differentialpattern of staining with M89 suggests Case #6 may have a distinctetiology and that it may be possible to distinguish these disease formsby immunoreactivity using conformational monoclonals.

Anti-Annular Protofibril Monoclonal antibodies, 09 and 89

Example 5 Antibody Production

Two New Zealand white rabbits were vaccinated with an antigen consistingof A131-40 carboxyl terminal thioester covalently bonded to colloidalgold particles via the carbosyl terminal sulfur atom. One hundredmicrograms of the peptide conjugate antigen was injected with incompleteFreunds adjuvant and boosted with the same antigen at 3 week intervalsfor approximately 5 months. After the immune response was determined tobe equivalent to the A11 antibody immune response, one of the animalswas sacrificed, the spleen harvested and the splenic lymphocytes used toproduce hybridomas via standard methods known in the art.

After culturing the hybridomas for a sufficient period of time, thesupernatants from multiclone wells were screened for the presence ofconformation dependent prefibrillar oligomer specific antibodies usingAβ prefibrillar oligomers as a primary screening agent. Aβ monomer,prefibrillar oligomers and fibrils were used as a secondary means ofexcluding antibodies that are not conformation dependent and interactwith all Aβ conformations. Approximately 118 multiclone wells wereselected as having immunoreactivity above a criterion of 0.5 AU. Thesemulti clones were sub-cloned and the resulting monoclones were subjectedto additional screening and characterization using Aβ40 monomer,prefibrillar oligomers and fibrils.

After secondary screening, clones having a distinct preference foreither prefibrillar oligomers or fibrils were selected. Representativeclones were selected in comparison to the A11 polyclonal antibody and6E10, a sequence dependent mouse monoclonal antibody. The conformationaland sequence specificity of the clones was analyzed by dot blot. Dotblot analysis was conducted by spotting 1 ug of A1340 monomer,prefibrillar oligomers and fibrils and 1 ug of prefibrillar oligomers ofalpha synuclein, immunoglobulin light chain, prion 106-126 peptide,KK(Q40)KK and calcitonin. A11 polyclonal antibody reacts with all typesof prefibrillar oligomers, but not Aβ monomer or fibrils. 6E10recognizes only samples containing Aβ. Clones 118, 201, 204, 205 and 206are specific for prefibrillar oligomers and do not recognize monomer orfibrils. This group of clones displays distinct specificities in termsof the other types of prefibrillar oligomers recognized. Clone 121 isspecific for A13 fibrils and does not recognize prefibrillar oligomersof any type or A13 monomer.

A number of clones appear to secrete antibodies of identicalspecificity. The most abundant class is similar to clone 201, which onlyrecognize Aβ prefibrillar oligomers and not oligomers of other types.All of these clones are also IgMs. Clone 118 is also indistinguishablefrom clones 48 and 55 (data not shown).

The sequences of two of the monoclonal IgGs, 118 and 204 are shownbelow. The amino acid sequences of the variable regions is distinct,consistent with their different specificities.

#118 kappa Vl + Cl (SEQ ID NO. 7)AQAAELVMTQTPASVSAAVGGTVTINCQSSESVYNSRLSWFQQKPGQPPKLLIYFASTLASGVSSRFSGSGSGTEFTLTISGVQCDDAATYYCAGHFSNSVYTFGGGTEVVVTGDPVAPTVLIFPPSADLVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCK VTQGTTSVVQSFNRGDC*#118 Vh + C1h (SEQ ID NO. 8)AAQPAMAQSVEESGGRLVTPGTPLTLTCTVSGFSLSAYEVSWVRQAPGKGLEWIGIIYANGNTVYASWAKGRFTISKTSTKVDLRIPSPTTEDTATYFCARDIYTTTTNLWGPGTLVTVSSGQPKAPSVFPLAPCCGDTHSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKTSC #204 kappa Vl + C1l (SEQ ID NO. 9)AQAAELDMTQTPASVSEPVGGTVTIKCQASQSISSYLAWYQQKPGQRPRLLIYETSTLASGVPSRFKGSGSGTEFTLTISDLECADAATYYCQSTYENPTYVSFGGGTEVGVKGDPVAPTVLIFPPSADLVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCK VTQGTTSVVQSFNRGDC*#204 Vh + C1h (SEQ ID NO. 10)AAQPAMAQSVKESGGRLVTPGTPLTLACTVSGFSLNTYSMFWVRQAPGKGLQWIGIISNFGVIYYATWAKGRFTISKTSTTVDLKITSPTTEDTATYFCVRKYGSEWGGDLWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKTSC #205 kappa Vl + C1l (SEQ ID NO. 11)AQAAELVMTQTPSSVSAAVGGTVTISCQSSESVYNNNYLSWYQQKPGQPPKRLIDSASTLDSGVPSRFKGSGSGAQFTLTISDLECDDAATYYCAGAYVNWMRIFGGGTEVVVKGDPVAPTVLIFPPSADLVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTC KVTQGTTSVVQSFNRGDC*#205 Vh + C1h (SEQ ID NO. 12)MAQSVEESGGRLVTPGTPLTLTCTASGFSLINYYMNWVRQAPGKGLEWIGLITGWADTYYANSAKGRFTISKTSSTTVDLKITSPTTDDTATYFCVRGGHTNIISLWGPGTLVTVSSGQPKAPSVFPLPPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRIFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKTSC

Monoclonal Antibodies Specific for Ala Oligomers and Fibrils:

Even though the polyclonal immune response to different assembly statesof amyloid is remarkably specific, monoclonal antibodies offer uniqueadvantages in terms of defining fine structural variation in amyloidaggregates and for determining the structures of these aggregates andtheir pathological significance. We initially focused on antibodiesagainst prefibrillar amyloid epitopes, but vaccinating mice with thecolloidal gold coupled Aβ that we used to make A11. We tried to producemouse monoclonal antibodies by contract with two different vendors using4 different strains of mice. Even though we obtained good titers ofconformation dependent IgGs in the polyclonal mouse serum, we were onlyable to clone IgM secreting hybridomas from the fusion products of mousesplenocytes. We tried different routes of vaccination, vaccination for 6months and different sources of lymphocytes (peripheral and lymph node)but were never able to clone anything but IgMs. The reasons for thefailure to clone IgG secreting clones from mouse hybridomas remainunclear. While these IgMs may have some utility, they are not asdesirable for many applications, so we contracted to make monoclonalIgGs in rabbits by contracting with Epitomics, Inc. Although we used thesame antigens and screening that we used for mouse monoclonals, weobtained many more positive independent clones (>200), many of which areIgGs (data not shown). Many of these clones appear to be phenotypicallyidentical and fall in to one of 6 distinct classes that we haveidentified so far (FIG. 1).

The specificity of the monoclonal antibodies we obtained is interestingfor several reasons (FIG. 1). All of the monoclonals obtained inresponse to vaccination with the A11 Aβ C-terminal thioester colloidalgold antigen are conformation specific even though we selected allclones that reacted with Aβ monomer, oligomers and fibrils. None of theclones recognize monomer like 6E10. This indicates that the immuneresponse to the solid phase antigen is highly conformation specific.None of the antibodies recognize both pure fibril and pure oligomersamples, indicating that the distribution of these epitopes is mutuallyexclusive. Secondly, most of the antibodies (M118, M204, M206, M206)recognize “generic epitopes” that are distributed on prefibrillaroligomers produced from other protein and peptide sequences. However,within this class of antibodies that recognize “generic” prefibrillaroligomer epitopes there is considerable variation in the types ofoligomers that the antibody recognizes. All of the generic monoclonalsrecognize Aβ oligomers because they were used as the primary screen, buteach antibody has a specificity more restricted than the A11 polyclonalimmune response. For example, M204 strongly recognizes most types ofoligomers, but it is distinctly less reactive with immunoglobulin lightchain oligomers. M205 reacts strongly with alpha synuclein and lightchain oligomers, but does not react will with prion 106-126, polyQ andcalcitonin oligomers. M118 prefers light chain and polyQ oligomers, butnot synuclein, prion or calcitonin oligomers. These results indicatethat there are multiple distinct epitopes associated with prefibrillaroligomers that are widely distributed within this class and thatmonoclonal antibodies can recognize these unique epitopes. Thirdly, somemonoclonals are both conformation dependent and sequence specific. M201recognizes only A13 oligomers, while M121 only recognizes A13 fibrils.M118, M204 and M205 are IgG, while the other antibodies are IgM.

Example 6

Monoclonals were made in rabbits under contract with Epitomics, Inc. NewZealand white rabbits were immunized with Aβ42 annular protofibrilsprepared as described in Kayed et al., J. Biol. Chem. 2009 (1).Homogenous populations of Aβ annular protofibrils were prepared by usingAβ prefibrillar oligomers as the starting material that were prepared aspreviously described (2). APFs were prepared by adding 5% (vol/vol)hexane to the solution of prefibrillar oligomers and the sample wasmixed with a vortex mixer for 1 min every 5 min for a total 50 min.Afterwards, the samples were dialyzed in water, using a MW cut-offmembrane of 10 kDa. Rabbits were vaccinated a total of 7 times at 3 weekintervals with 500 ug of Aβ annular protofibrils. The serum was screenedfor annular protofibril specific titer and the rabbit with the highesttiter was chosen for monoclonal production. The supernatants from theresulting hybridomas were initially screened by ELISA with Aβ annularprotofibrils, Aβ fibrils and Aβ monomer. Wells that produced an opticaldensity of greater than 1.0 on annular protofibrils and backgroundreactivity on Aβ fibrils and monomer were chosen for secondaryscreening. Approximately 100 wells were chosen for secondary screeningagainst Aβ annular protofibrils (APFs), alpha hemolysin pores, Aβfibrils, Aβ prefibrillar oligomers, Aβ monomer and Aβ in 0.1% SDS usingdot blotting. The results are shown below.

In the secondary screen, monoclonals 09 and 89 only reacted with thealpha hemolysin pores. We also characterized their immunoreactivityagainst alpha hemolysin and Aβ dissolved in 10 mM NaOH by westernblotting as shown below.

Both monoclonal antibodies 09 and 87 were found to react with a band atapproximately 65 kDa on Westerns. M87 also reacts with a band atapproximately 40 kDa. The band of alpha hemolysin at 65 kDa also reactsstrongly with the polyclonal serum from the rabbit that the monoclonalswere prepared from.

-   1. Kayed, R., A. Pensalfini, L. Margol, Y. Sokolov, F. Sarsoza, E.    Head, J. Hall, and C. Glabe. 2009. Annular protofibrils are a    structurally and functionally distinct type of amyloid oligomer. J    Biol Chem 284:4230-4237.-   2. Kayed, R., and C. G. Glabe. 2006. Conformation-dependent    anti-amyloid oligomer antibodies. Methods Enzymol 413:326-344.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of cell biology, cell culture,molecular biology, microbiology, recombinant DNA, and immunology, whichare within the skill of the art. Such techniques are described in theliterature.

This invention is not limited in its application to the details ofconstruction and the arrangement of components set forth in thedescription or illustrated in the drawings. The invention is capable ofother embodiments and of being practiced or of being carried out invarious ways. The phraseology and terminology used herein is for thepurpose of description and should not be regarded as limiting. The useof “including,” “comprising,” or “having,” “containing,” “involving,”and variations thereof herein, is meant to encompass the items listedthereafter and equivalents thereof as well as additional items.

1.-57. (canceled)
 58. A method, comprising: (a) applying to an area of asurface a solution comprising (i) urine from a pregnant woman and (ii)dye, wherein the dye is selected from Congo Red, Thioflavin S,Thioflavin T, Evans Blue, Trypan blue, amino-8-napthalene sulfonate(ANS), and bis-azo ANS; and (b) assessing staining intensity of the dyein the area relative to a positive control or a negative control. 59.The method of claim 58, wherein the surface comprises nitrocellulose.60. The method of claim 58, wherein the urine comprises proteinaggregates.
 61. The method of claim 58, further comprising identifyingthe urine as having protein aggregates if the staining intensity of thedye is greater than a negative control or comparable to a positivecontrol.
 62. The method of claim 61, wherein the pregnant woman haspreeclampsia.
 63. The method of claim 61, wherein the pregnant woman isat risk of preeclampsia.
 64. The method of claim 58, wherein thestaining intensity of the dye is assessed by a dot blot assay.
 65. Themethod of claim 58, wherein the staining intensity of the dye isassessed by a spectral shift assay.
 66. The method of claim 58, whereinthe dye is Congo Red.
 67. The method of claim 58, wherein the dye isThioflavin S or Thioflavin T.
 68. A method, comprising: (a) applying toan area of a surface a solution comprising (i) urine from a pregnantwoman and (ii) Congo Red dye; and (b) assessing staining intensity ofthe Congo Red dye in the area relative to a positive control or anegative control.
 69. The method of claim 68, wherein the surfacecomprises nitrocellulose.
 70. The method of claim 68, wherein the urinecomprises protein aggregates.
 71. The method of claim 68, furthercomprising identifying the urine as having protein aggregates if thestaining intensity of the dye is greater than a negative control orcomparable to a positive control.
 72. The method of claim 71, whereinthe pregnant woman has preeclampsia.
 73. The method of claim 71, whereinthe pregnant woman is at risk of preeclampsia.
 74. The method of claim68, wherein the staining intensity of the dye is assessed by a dot blotassay.
 75. The method of claim 68, wherein the staining intensity of thedye is assessed by a spectral shift assay.
 76. A method, comprising: (a)applying to an area of a nitrocellulose surface a solution comprising(i) urine from a pregnant woman and (ii) Congo Red dye; and (b)assessing staining intensity of the Congo Red dye in the area relativeto a positive control or a negative control.
 77. The method of claim 76,further comprising identifying the urine as having protein aggregates ifthe staining intensity of the dye is greater than a negative control orcomparable to a positive control.